Analysis of glutamate receptor surface expression in acute hippocampal slices.

Trafficking of receptors to and from the cell surface is a powerful mechanism for regulating neuronal excitability. To date, the majority of studies concerning glutamate receptor trafficking have been performed in neuronal cultures in which surface expression can be readily assayed by immunofluorescence techniques. Results from such studies have had important implications in the field of synaptic plasticity. However, cultured neurons are, by necessity, prepared from very young animals. Moreover, although an enhancement of excitatory neurotransmission can be induced in such systems, classic long-term potentiation (LTP) can be produced only in acute slices or in vivo. To study trafficking in adult tissues, we have adapted two biochemical techniques, proteolysis and cross-linking. These techniques help define surface-expressed and intracellular pools of native receptors in acute hippocampal slices.

LTP leads to rapid surface expression of NMDA but not AMPA receptors in adult rat CA1.

In the CA1 region of the rat hippocampus, long-term potentiation (LTP) requires the activation of NMDA receptors (NMDARs) and leads to an enhancement of AMPA receptor (AMPAR) function. In neonatal hippocampus, this increase in synaptic strength seems to be mediated by delivery of AMPARs to the synapse. Here we studied changes in surface expression of native AMPA and NMDA receptors following induction of LTP in the adult rat brain. In contrast to early postnatal rats, we find that LTP in the adult rat does not alter membrane association of AMPARs. Instead, LTP leads to rapid surface expression of NMDARs in a PKC- and Src-family-dependent manner. The present study suggests a developmental shift in the LTP-dependent trafficking of AMPA receptors. Moreover, our results indicate that insertion of NMDA receptors may be a key step in regulating synaptic plasticity.

Deficits in the expression of the NR2B subunit in the hippocampus of aged Fisher 344 rats.

The NMDA receptor (NMDAR) has been implicated in the induction of LTP at hippocampal synapses, and has been proposed to play a significant role in the involvement of the hippocampus with learning and memory. Aged rats are known to have deficits in LTP, learning and memory. We tested the hypothesis that aged rats might have deficits in expression of NMDAR subunits. Aged rats have significantly lower levels of NR2B mRNA and protein compared to young animals. This complements a recent report which showed improved learning and memory in mice which overexpress NR2B. No changes were seen in either the mRNA or the protein levels of the NMDAR subunit NR2A, nor in the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate receptor (AMPAR) subunit GluR2. Our data support the hypothesis that age related alterations in the expression of the NMDAR NR2B subunit might underlie deficits in LTP and learning and memory in aged animals.

Transcription and replication of mitochondrial DNA.

The physical isolation of mammalian mitochondrial DNA (mtDNA) over 30 years ago marked the beginning of studies of its structure, replication and the expression of its genetic content. Such analyses have revealed a number of surprises: novel DNA structural features of the circular genome such as the displacement loop (D-loop); multiple sized and deleted forms of the circular genome; a minimal set of mitochondrially encoded rRNAs and tRNAs needed for translation; a bacteriophage-like, nuclear-encoded mitochondrial RNA polymerase for transcription; and a direct linkage between transcription and the commitment to replication of the leading mtDNA strand that centres on the nuclear encoded mitochondrial transcription factor A. One of the more recent revelations is the existence, near the D-loop, of an atypical, stable RNA-DNA hybrid (or R-loop) at the origin of mammalian leading-strand DNA replication, composed of the parent DNA strands and an RNA transcript. In mammalian mitochondrial systems, all of the proteins known to be involved in DNA replication are encoded in the nucleus. Thus alterations and deficiencies in mtDNA replication must arise from mutations in mtDNA regulatory sequences and nuclear gene defects. Further studies of the relationships between nuclear-encoded proteins and their mtDNA target sequences could result in strategies to manipulate genotypes within cellular mtDNA populations.

Mutational analysis of the RNA component of Saccharomyces cerevisiae RNase MRP reveals distinct nuclear phenotypes.

The 340-nucleotide RNA component of Saccharomyces cerevisiae RNase MRP is encoded by the single-copy essential gene, NME1. To gain additional insight into the proposed structure and functions of this endoribonuclease, we have extensively mutagenized the NME1 gene and characterized yeast strains expressing mutated forms of the RNA using a gene shuffle technique. Strains expressing each of 26 independent mutations in the RNase MRP RNA gene were characterized for their ability to grow at various temperatures and on various carbon sources, stability of the RNase MRP RNA and processing of the 5.8S rRNA (a nuclear function of RNase MRP). 11 of the mutations resulted in a lethal phenotype, six displayed temperature-conditional lethality, and several preferred a non-fermentable carbon source for growth. In those mutants that exhibited altered growth phenotypes, the severity of the growth defect was directly proportional to the severity of the 5.8S rRNA processing defect in the nucleus. Together this analysis has defined essential regions of the RNase MRP RNA and provides evidence that is consistent with the proposed function of the RNase MRP enzyme.

Vertebrate mitochondrial DNA-a circle of surprises.

Evidence for the existence of a vertebrate mitochondrial genome first arose over 30 years ago. Application of emerging techniques of molecular biology established the structure of vertebrate mitochondrial DNA (mtDNA) as a small closed-circular species. The ability to purify these mtDNAs to a high degree facilitated studies on the overall replication and expression pattern of the genome. With the acquisition of the genomic sequences of human and mouse mtDNAs, it was possible to infer the genetic organization and some of the genes contained therein, as well as providing a basis for developing strategies to assign important regulatory elements involved in mtDNA replication and transcription. This, in turn, presented the opportunity to identify nucleus-encoded proteins that target to mtDNA and, in doing so, determine the replication and expression modes of the genome. Vertebrate cells, in general, need mtDNA due to the requirements for maintaining a functional oxidative phosphorylation pathway. Depression of mtDNA content or mutations in mtDNA can result in metabolic dysfunction severe enough, in some cases, to result in human lethality. The emergence of mouse models for human mitochondrial diseases should provide the experimental context to understand the full role of mtDNA in different cells, tissues, and organs; the control of organelle biogenesis; and the development of therapeutic strategies for treatment of mitochondrial disorders.

Mitochondrial medicine--recent advances.

Mitochondria contain the respiratory chain enzyme complexes that carry out oxidative phosphorylation and produce the main part of cellular energy in the form of ATP. Mitochondrial DNA (mtDNA) encodes essential subunits of the respiratory chain and is thus critical for maintaining cellular energy production. The first pathogenic mtDNA mutations were reported in 1988, and today more than 50 disease-causing mtDNA mutations have been identified. In addition, mtDNA mutations have been implicated in ageing and in common disorders such as diabetes mellitus, heart failure and Parkinson's disease. This review will summarize recent advances in the rapidly expanding field of mitochondrial medicine.

Dilated cardiomyopathy and atrioventricular conduction blocks induced by heart-specific inactivation of mitochondrial DNA gene expression.

Mutations of mitochondrial DNA (mtDNA) cause several well-recognized human genetic syndromes with deficient oxidative phosphorylation and may also have a role in ageing and acquired diseases of old age. We report here that hallmarks of mtDNA mutation disorders can be reproduced in the mouse using a conditional mutation strategy to manipulate the expression of the gene encoding mitochondrial transcription factor A (Tfam, previously named mtTFA), which regulates transcription and replication of mtDNA. Using a loxP-flanked Tfam allele (TfamloxP) in combination with a cre-recombinase transgene under control of the muscle creatinine kinase promoter, we have disrupted Tfam in heart and muscle. Mutant animals develop a mosaic cardiac-specific progressive respiratory chain deficiency, dilated cardiomyopathy, atrioventricular heart conduction blocks and die at 2-4 weeks of age. This animal model reproduces biochemical, morphological and physiological features of the dilated cardiomyopathy of Kearns-Sayre syndrome. Furthermore, our findings provide genetic evidence that the respiratory chain is critical for normal heart function.

cDNA cloning, sequencing, and characterization of male and female rat liver aldehyde oxidase (rAOX1). Differences in redox status may distinguish male and female forms of hepatic APX.

Molecular characterization of male and female rat liver aldehyde oxidase is reported. As described for the mouse liver, male and female rat liver expressed kinetically distinct forms of aldehyde oxidase. Our data suggest that the two forms arise as a result of differences in redox state and are most simply explained by expression of a single gene encoding aldehyde oxidase in rats. In support of this argument we have sequenced cDNAs from male and female rat liver. We examined mRNA expression by Northern blot analysis with RNA from males and females, from several tissues, and following androgen induction. Purified rat liver enzyme from males or females revealed a single 150-kDa species consistent with cDNA sequence analysis. Both male and female forms were reactive to the same carboxyl-terminal directed antisera. Km(app) values obtained in crude extracts of male or female rat liver and post-benzamidine-purified aldehyde oxidase differed substantially from each other but could be interconverted by chemical reduction with dithiothreitol or oxidation with 4,4'-dithiodipyridine. Our data indicate that a single gene is most likely expressed in male or female rat liver and that the kinetic differences between male and female rat liver aldehyde oxidases are sensitive to redox manipulation.

Stimulation of mitochondrial gene expression and proliferation of mitochondria following impairment of cellular energy transfer by inhibition of the phosphocreatine circuit in rat hearts.

Mitochondria proliferate when cellular energy demand increases. However, the pathways leading to enhanced expression of mitochondrial genes are largely unknown. We tested the hypothesis that an altered flux through energy metabolism is the key regulatory event by decreasing mitochondrial energy supply to rat heart cells by creatine depletion. Electron microscopy showed that the density of mitochondria increased by 75% in such hearts (p < 0.01). Levels of representative mRNAs encoded on mitochondrial DNA (mtDNA) or on nuclear chromosomes were elevated 1.5 to 2-fold (p < 0.05), while the mtDNA content was unchanged. The mRNA for the nuclear encoded mitochondrial transcription factor A (mtTFA) was increased after GPA feeding (p < 0.05). Thus, we have shown that an impairment of mitochondrial energy supply causes stimulation of gene expression resulting in mitochondrial proliferation, probably as a compensatory mechanism. The observed activation of the mtTFA gene corroborates the important function of this protein in nuclear-mitochondrial communication.

Initiation of mitochondrial DNA replication by transcription and R-loop processing.

The mitochondrial genome of eukaryotic cells is maintained by a mechanism distinct from that employed in the nucleus. Mitochondrial DNA replication at the leading-strand origin is coupled to transcription through the formation of an RNA-DNA hybrid known as an R-loop. In vivo and in vitro evidence has implicated an RNA processing enzyme, RNase MRP, in primer maturation. In our investigation of mammalian RNase MRP, we have analyzed its specific endoribonuclease activity on model R-loops. We demonstrate here that human RNase MRP cleaves this distinctly configured substrate at virtually all of the major DNA replication sites previously mapped in vivo. We further show that the processed RNA products remain stably base-paired to the template DNA strand and are functional for initiating DNA synthesis on a closed circular plasmid. Thus, in vitro initiation of leading-strand mtDNA synthesis requires only the actions of RNA polymerase and RNase MRP for the generation of replication primers.

The gene for ribosomal protein L7a-1 in Schizosaccharomyces pombe contains an intron after the initiation codon.

The gene encoding ribosomal protein L7a-1 in the fission yeast Schizosaccharomyces pombe is identified by the similarity of its open reading frame to the respective gene in Saccharomyces cerevisiae. The L7a gene is encoded in two different genomic environments as frequently found for ribosomal protein genes in this organism. One of these genes, L75a-1, is located on chromosome 2. The two consensus promoter elements homol D and homol E are both identified upstream of the start codon of this gene. The ATG start codon is separated from the main reading frame by an intron of 66 nucleotides.

Transcription-dependent DNA transactions in the mitochondrial genome of a yeast hypersuppressive petite mutant.

Mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae contains highly conserved sequences, called rep/ori, that are associated with several aspects of its metabolism. These rep/ori sequences confer the transmission advantage exhibited by a class of deletion mutants called hypersuppressive petite mutants. In addition, because they share features with the mitochondrial leading-strand DNA replication origin of mammals, rep/ori sequences have also been proposed to participate in mtDNA replication initiation. Like the mammalian origins, where transcription is used as a priming mechanism for DNA synthesis, yeast rep/ori sequences contain an active promoter. Although transcription is required for maintenance of wild-type mtDNA in yeast, the role of the rep/ori promoter as a cis-acting element involved in the replication of wild-type mtDNA is unclear, since mitochondrial deletion mutants need neither transcription nor a rep/ori sequence to maintain their genome. Similarly, transcription from the rep/ori promoter does not seem to be necessary for biased inheritance of mtDNA. As a step to elucidate the function of the rep/ori promoter, we have attempted to detect transcription-dependent DNA transactions in the mtDNA of a hypersuppressive petite mutant. We have examined the mtDNA of the well-characterized petite mutant a-1/1R/Z1, whose repeat unit shelters the rep/ori sequence ori1, in strains carrying either wild-type or null alleles of the nuclear genes encoding the mitochondrial transcription apparatus. Complex DNA transactions were detected that take place around GC-cluster C, an evolutionarily conserved GC-rich sequence block immediately downstream from the rep/ori promoter. These transactions are strictly dependent upon mitochondrial transcription.

Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice.

The regulation of mitochondrial DNA (mtDNA) expression is crucial for mitochondrial biogenesis during development and differentiation. We have disrupted the mouse gene for mitochondrial transcription factor A (Tfam; formerly known as m-mtTFA) by gene targetting of loxP-sites followed by cre-mediated excision in vivo. Heterozygous knockout mice exhibit reduced mtDNA copy number and respiratory chain deficiency in heart. Homozygous knockout embryos exhibit a severe mtDNA depletion with abolished oxidative phosphorylation. Mutant embryos proceed through implantation and gastrulation, but die prior to embryonic day (E)10.5. Thus, Tfam is the first mammalian protein demonstrated to regulate mtDNA copy number in vivo and is essential for mitochondrial biogenesis and embryonic development.

Backfoot is a novel homeobox gene expressed in the mesenchyme of developing hind limb.

Homeobox genes play important roles in pattern formation during development. Here, we report the cloning and temporal and spatial expression patterns of a novel homeobox gene Backfoot (BFT for the human gene, and Bft for the mouse gene), whose expression reveals an early molecular distinction between forelimb and hind limb. BFT was identified as a sequence-specific DNA-binding protein. In addition to the homeodomain, it shares a carboxyl-terminal peptide motif with other paired-like homeodomain proteins. Northern hybridization analysis of RNAs from human tissues revealed that human BFT is highly expressed in adult skeletal muscle and bladder. During midgestation embryogenesis, mouse Bft is expressed in the developing hind limb buds, mandibular arches, and Rathke's pouch. The expression of Bft begins prior to the appearance of hind limb buds in mesenchyme but is never observed in forelimbs. At later stages of limb development, the expression is progressively restricted to perichondrial regions, most likely in tendons and ligaments. The timing and pattern of expression suggest that Bft plays multiple roles in hind limb patterning, branchial arch development, and pituitary development. Bft is likely identical to a mouse gene, Ptx1, that was recently isolated by Lamonerie et al. ([1996] Genes Dev. 10:1284-1295) and that has been suggested to play a role in pituitary development.

RNase mitochondrial RNA processing correctly cleaves a novel R loop at the mitochondrial DNA leading-strand origin of replication.

The precursor primer RNA for mammalian mitochondrial DNA leading-strand replication remains as a persistent R loop formed during transcription through the mitochondrial DNA control region. We have examined model R loops, which exist in a novel and physiologically accurate preprimer conformation, as potential substrates for mammalian RNase mitochondrial RNA processing (MRP). Mouse RNase MRP accurately cleaves an R loop containing the mouse mitochondrial DNA origin. The multiple cleavage sites on the R-loop substrate match the priming sites observed in vivo, suggesting that RNase MRP alone is capable of generating virtually all of the leading-strand replication primers.

Down-regulation of mitochondrial transcription factor A during spermatogenesis in humans.

Mitochondrial transcription factor A (mtTFA) is a key activator of mitochondrial transcription in mammals. It also has a role in mitochondrial DNA (mtDNA) replication, since transcription generates an RNA primer necessary for initiation of mtDNA replication. In the mouse, testis-specific mtTFA transcripts encode a protein isoform that is imported to the nucleus rather than into mitochondria of spermatocytes and elongating spermatids. We now report molecular characterization of human mtTFA (h-mtTFA) expression in somatic tissues and male germ cells. Similarly to the mouse, analysis of cDNAs and Northern blots identified abundant testis-specific transcript isoforms generated by use of alternate transcription initiation sites. However, unlike the mouse, none of the testis-specific transcripts predicts a nuclear protein isoform, and Western blot analysis identified only the mitochondrial form of h-mtTFA in human testis. Immunohistochemistry and in situ were used to compare the distribution of mtTFA protein, testis-specific mtTFA transcripts, mtDNA and mtRNA in sections of human testis. Our results show that the mtTFA protein and mtDNA exhibit parallel gradients with high levels in undifferentiated male germ cells and low levels or an absence in different male germ cells. Testis-specific transcripts exhibit the opposite pattern, suggesting that in both humans and mice, these testis-specific mtTFA transcripts down-regulate mtTFA protein levels in mammalian mitochondria. Our findings demonstrate that mtTFA does not have a critical role in the nucleus, suggest a mechanism for reducing mtDNA copy number during spermatogenesis and have implications for the understanding of maternal transmission of mtDNA.