Humanin, a newly identified neuroprotective factor, uses the G protein-coupled formylpeptide receptor-like-1 as a functional receptor

Alzheimer's disease (AD) is characterized by overproduction of beta amyloid peptides in the brain with progressive loss of neuronal cells. The 42-aa form of the beta amyloid peptide (Abeta(42)) is implied as a major causative factor, because it is toxic to neurons and elicits inflammatory responses in the brain by activating microglial cells. Despite the overproduction of Abeta(42), AD brain tissue also generates protective factor(s) that may antagonize the neurodestructive effect of Abeta(42). Humanin is a gene cloned from an apparently normal region of an AD brain and encodes a 24-aa peptide. Both secreted and synthetic Humanin peptides protect neuronal cells from damage by Abeta(42), and the effect of Humanin may involve putative cellular receptor(s). To elucidate the molecular identity of such receptor(s), we examined the activity of synthetic Humanin on various cells and found that Humanin induced chemotaxis of mononuclear phagocytes by using a human G protein-coupled formylpeptide receptor-like-1 (FPRL1) and its murine counterpart FPR2. Coincidentally, FPRL1 and FPR2 are also functional receptors used by Abeta(42) to chemoattract and activate phagocytic cells. Humanin reduced the aggregation and fibrillary formation by suppressing the effect of Abeta(42) on mononuclear phagocytes. In neuroblast cells, Humanin and Abeta(42) both activated FPRL1; however, only Abeta(42) caused apoptotic death of the cells, and its cytopathic effect was blocked by Humanin. We conclude that Humanin shares human FPRL1 and mouse FPR2 with Abeta(42) and suggest that Humanin may exert its neuroprotective effects by competitively inhibiting the access of FPRL1 to Abeta(42).     

Cell and animal models of mtDNA biology: progress and prospects

The past two decades have witnessed an evolving understanding of the mitochondrial genome’s (mtDNA) role in basic biology and disease. From the recognition that mutations in mtDNA can be responsible for human disease to recent efforts showing that mtDNA mutations accumulate over time and may be responsible for some phenotypes of aging, the field of mitochondrial genetics has greatly benefited from the creation of cell and animal models of mtDNA mutation. In this review, we critically discuss the past two decades of efforts and insights gained from cell and animal models of mtDNA mutation. We attempt to reconcile the varied and at times contradictory findings by highlighting the various methodologies employed and using human mtDNA disease as a guide to better understanding of cell and animal mtDNA models. We end with a discussion of scientific and therapeutic challenges and prospects for the future of mtDNA transfection and gene therapy. mitochondria; mitochondrial DNA; cybrid     

Activity-dependent neurotrophic factor, ADNF, determines the structure characteristics of Colivelin, a fusion protein of ADNF9 and Humanin analog.

A 24-amino acid long peptide, Humanin, protects neurons from Alzheimer's disease (AD)-related cell toxicities at sub-nM-uM concentrations. Activity-dependent neurotrophic factor (ADNF) is a glia-derived neurotrophic peptide, which protects neurons from tetrodoxin treatment and AD-related and amyotrophic lateral sclerosis-related insults at fM concentrations. An attempt was made to further improve the activity of Humanin by fusing this peptide to ADNF9, a 9-amino acid long core peptide of the ADNF. This fusion resulted in a novel molecule, termed Colivelin, with the neuroprotective activity at fM range, which is approximately 10(3)-10(7) fold higher than the activity of Humanin and Humanin analogs and follows the activity profile of fM-active ADNF9. We have characterized the structural properties of Colivelin and compared with those of ADNF9 and Humanin in water and phosphate-buffered saline (PBS). The secondary structure of Colivelin was similar to that of ADNF9, but not that of Humanin, and hence was not the average of the contributions of the two peptides fused. Colivelin was stable and monomeric in PBS, consistent with the monomeric property of ADNF9, while Humanin showed strong tendency to self-associate. Thus, it is evident that the structural properties of Colivelin resemble those of ADNF9, rather than those of Humanin.     

The complete nucleotide sequence of a snake (Dinodon semicarinatus) mitochondrial genome with two identical control regions.

The 17,191-bp mitochondrial DNA (mtDNA) of a Japanese colubrid snake, akamata (Dinodon semicarinatus), was cloned and sequenced. The snake mtDNA has some peculiar features that were found in our previous study using polymerase chain reaction: duplicate control regions that have completely identical sequences over 1 kbp, translocation of tRNALeu(UUR) gene, shortened TpsiC arm for most tRNA genes, and a pseudogene for tRNAPro. Phylogenetic analysis of amino acid sequences of protein genes suggested an unusually high rate of molecular evolution in the snake compared to other vertebrates. Southern hybridization experiments using mtDNAs purified from multiple akamata individuals showed that the duplicate state of the control region is not a transient or unstable feature found in a particular individual, but that it stably occurs in mitochondrial genomes of the species. This may, therefore, be regarded as an unprecedented example of stable functional redundancy in animal mtDNA. However, some of the examined individuals contain a rather scanty proportion of heteroplasmic mtDNAs with an organization of genes distinct from that of the major mtDNA. The gene organization of the minor mtDNA is in agreement with one of models that we present to account for the concerted evolution of duplicate control regions.     

A method for mutagenesis of mouse mtDNA and a resource of mouse mtDNA mutations for modeling human pathological conditions

Mutations in human mitochondrial DNA (mtDNA) can cause mitochondrial disease and have been associated with neurodegenerative disorders, cancer, diabetes and aging. Yet our progress toward delineating the precise contributions of mtDNA mutations to these conditions is impeded by the limited availability of faithful transmitochondrial animal models. Here, we report a method for the isolation of mutations in mouse mtDNA and its implementation for the generation of a collection of over 150 cell lines suitable for the production of transmitochondrial mice. This method is based on the limited mutagenesis of mtDNA by proofreading-deficient DNA-polymerase γ followed by segregation of the resulting highly heteroplasmic mtDNA population by means of intracellular cloning. Among generated cell lines, we identify nine which carry mutations affecting the same amino acid or nucleotide positions as in human disease, including a mutation in the ND4 gene responsible for 70% of Leber Hereditary Optic Neuropathies (LHON). Similar to their human counterparts, cybrids carrying the homoplasmic mouse LHON mutation demonstrated reduced respiration, reduced ATP content and elevated production of mitochondrial reactive oxygen species (ROS). The generated resource of mouse mtDNA mutants will be useful both in modeling human mitochondrial disease and in understanding the mechanisms of ROS production mediated by mutations in mtDNA.     

Secreted calmodulin-like skin protein inhibits neuronal death in cell-based Alzheimer's disease models via the heterotrimeric Humanin receptor

Humanin is a secreted bioactive peptide that is protective in a variety of death models, including cell-based neuronal death models related to Alzheimer''s disease (AD). To mediate the protective effect in AD-related death models, Humanin signals via a cell-surface receptor that is generally composed of three subunits: ciliary neurotrophic factor receptor α, WSX-1 and gp130 (heterotrimeric Humanin receptor; htHNR). However, the protective effect of Humanin via the htHNR is weak (EC₅₀=1–10 μℳ); therefore, it is possible that another physiological agonist for this receptor exists in vivo. In the current study, calmodulin-like skin protein (CLSP), a calmodulin relative with an undefined function, was shown to be secreted and inhibit neuronal death via the htHNR with an EC₅₀ of 10–100 pℳ. CLSP was highly expressed in the skin, and the concentration in circulating normal human blood was ∼5 nℳ. When administered intraperitoneally in mice, recombinant CLSP was transported across the blood-cerebrospinal fluid (CSF)-barrier and its concentration in the CSF reaches 1/100 of its serum concentration at 1 h after injection. These findings suggest that CLSP is a physiological htHNR agonist.     

Comparison between the complete mtDNA sequences of the blue and the fin whale,two species that can hybridize in nature

The sequence of the mitochondrial DNA (mtDNA) molecule of the blue whale (Balaenoptera musculus) was determined. The molecule is 16,402 by long and its organization conforms with that of other eutherian mammals. The molecule was compared with the mtDNA of the congeneric fin whale (B. physalus). It was recently documented that the two species can hybridize and that male offspring are infertile whereas female offspring may be fertile. The present comparison made it possible to determine the degree of mtDNA difference that occurs between two species that are not completely separated by hybridization incompatibility. The difference between the complete mtDNA sequences was 7.4%. Lengths of peptide coding genes were the same in both species. Except for a small portion of the control region, disruption in alignment was usually limited to insertion/deletion of a single nucleotide. Nucleotide differences between peptide coding genes ranged from 7.1 to 10.5%, and difference at the inferred amino acid level was 0.0–7.9%. In the rRNA genes the mean transition difference was 3.8%. This figure is similar in degree to the difference (3.4%) between the 12S rRNA gene of humans and the chimpanzee. The mtDNA differences between the two whale species, involving both peptide coding and rRNA genes, suggest an evolutionary separation of 5 million years. Although hybridization between more distantly related mammalian species may not be excluded, it is probable that the blue and fin whales are nearly as different in their mtDNA sequences as hybridizing mammal species may be. Correspondence to: Ú. Árnason     

Evidence for recombination of mtDNA in the marine mussel Mytilus trossulus from the Baltic

A number of studies have claimed that recombination occurs in animal mtDNA, although this evidence is controversial. Ladoukakis and Zouros (2001) provided strong evidence for mtDNA recombination in the COIII gene in gonadal tissue in the marine mussel Mytilus galloprovincialis from the Black Sea. The recombinant molecules they reported had not however become established in the population from which experimental animals were sampled. In the present study, we provide further evidence of the generality of mtDNA recombination in Mytilus by reporting recombinant mtDNA molecules in a related mussel species, Mytilus trossulus, from the Baltic. The mtDNA region studied begins in the 16S rRNA gene and terminates in the cytochrome b gene and includes a major noncoding region that may be analogous to the D-loop region observed in other animals. Many bivalve species, including some Mytilus species, are unusual in that they have two mtDNA genomes, one of which is inherited maternally (F genome) the other inherited paternally (M genome). Two recombinant variants reported in the present study have population frequencies of 5% and 36% and appear to be mosaic for F-like and M-like sequences. However, both variants have the noncoding region from the M genome, and both are transmitted to sperm like the M genome. We speculate that acquisition of the noncoding region by the recombinant molecules has conferred a paternal role on mtDNA genomes that otherwise resemble the F genome in sequence.     

The secondary structure analysis of a potent Ser14Gly analog of antiAlzheimer peptide, Humanin, by circular dichroism.

The structure of a highly potent Ser14Gly analog of antiAlzheimer peptide, Humanin, was examined by circular dichroism (CD). The secondary structure is more disordered in water than in phosphate-buffered saline (PBS). The peptide structure in water is little dependent on both peptide concentration and temperature. On the contrary, the peptide structure was significantly different in PBS from the structure in water, which is more apparent at a higher peptide concentration and temperature. The observed different structure in PBS appears to be due to self-association of the peptide, which is enhanced by elevated temperature and, hence, via hydrophobic interactions. The wild-type Humanin also behaved similarly, i.e., it assumed a disordered structure in water but underwent conformational changes in PBS. Although high peptide concentrations for CD measurements are not encountered in vivo, the results suggest the tendency of the peptide to interact hydrophobically with other structures as well as with itself.     

Humanin and the Receptors for Humanin

Alzheimer’s disease (AD) is a prevalent dementia-causing neurodegenerative disease. Neuronal death is closely linked to the progression of AD-associated dementia. Accumulating evidence has established that a 24-amino-acid bioactive peptide, Humanin, protects neurons from AD-related neuronal death. A series of studies using various murine AD models including familial AD gene-expressing transgenic mice have shown that Humanin is effective against AD-related neuronal dysfunction in vivo. Most recently, it has been shown that Humanin inhibits neuronal cell death and dysfunction by binding to a novel IL-6-receptor-related receptor(s) on the cell surface involving CNTFRα, WSX-1, and gp130. These findings suggest that endogenous Humanin [or a Humanin-like substance(s)] may suppress the onset of AD-related dementia by inhibiting both AD-related neuronal cell death and dysfunction.     

A broad survey of recombination in animal mitochondria

Recombination in mitochondrial DNA (mtDNA) remains a controversial topic. Here we present a survey of 279 animal mtDNA data sets, of which 12 were from asexual species. Using four separate tests, we show that there is widespread evidence of recombination; for one test as many as 14.2% of the data sets reject a model of clonal inheritance and in several data sets, including primates, the recombinants can be identified visually. We show that none of the tests give significant results for obligate clonal species (apomictic pathogens) and that the sexual species show significantly greater evidence of recombination than asexual species. For some data sets, such as Macaca nemestrina, additional data sets suggest that the recombinants are not artifacts. For others, it cannot be determined whether the recombinants are real or produced by laboratory error. Either way, the results have important implications for how mtDNA is sequenced and used.     

Genetic Variation and Geographic Differentiation in Mitochondrial DNA of the Horseshoe Crab, LIMULUS POLYPHEMUS

Restriction site variation in mitochondrial DNA (mtDNA) of the horseshoe crab (Limulus polyphemus) was surveyed in populations ranging from New Hampshire to the Gulf Coast of Florida. MtDNA clonal diversity was moderately high, particularly in southern samples, and a major genetic "break" (nucleotide sequence divergence approximately 2%) distinguished all sampled individuals which were north vs. south of a region in northeastern Florida. The area of genotypic divergence in Limulus corresponds to a long-recognized zoogeographic boundary between warm-temperate and tropical marine faunas, and it suggests that selection pressures and/or gene flow barriers associated with water mass differences may also influence the evolution of species widely distributed across such transition zones. On the other hand, a comparison of the mtDNA divergence patterns in Limulus with computer models involving stochastic lineage extinction in species with limited gene flow demonstrates that deterministic explanations need not necessarily be invoked to account for the observations. Experiments to distinguish stochastic from deterministic possibilities are suggested. Overall, the pattern and magnitude of mtDNA differentiation in horseshoe crabs is very similar to that typically reported for freshwater and terrestrial species assayed over a comparable geographic range. Results demonstrate for the first time that, geographically, at least some continuously distributed marine organisms can show considerable mtDNA genetic differentiation.     

Drosophila chemoreceptor gene evolution: selection, specialization and genome size

Chemoperception plays a key role in adaptation and speciation in animals, and the senses of olfaction and gustation are mediated by gene families which show large variation in repertoire size among species. In Drosophila, there are around 60 loci of each type and it is thought that ecological specialization influences repertoire size, with increased pseudogenization of loci. Here, we analyse the size of the gustatory and olfactory repertoires among the genomes of 12 species of Drosophila . We find that repertoire size varies substantially and the loci are evolving by duplication and pseudogenization, with striking examples of lineage-specific duplication. Selection analyses imply that the majority of loci are subject to purifying selection, but this is less strong in gustatory loci and in loci prone to duplication. In contrast to some other studies, we find that few loci show statistically significant evidence of positive selection. Overall genome size is strongly correlated with the proportion of duplicated chemoreceptor loci, but genome size, specialization and endemism may be interrelated in their influence on repertoire size.     

Lineage-specific loss of function of bitter taste receptor genes in humans and nonhuman primates

Since the process of becoming dead genes or pseudogenes (pseudogenization) is irreversible and can occur rather rapidly under certain environmental circumstances, it is one plausible determinant for characterizing species specificity. To test this evolutionary hypothesis, we analyzed the tempo and mode of duplication and pseudogenization of bitter taste receptor (T2R) genes in humans as well as in 12 nonhuman primates. The results show that primates have accumulated more pseudogenes than mice after their separation from the common ancestor and that lineage-specific pseudogenization becomes more conspicuous in humans than in nonhuman primates. Although positive selection has operated on some amino acids in extracellular domains, functional constraints against T2R genes are more relaxed in primates than in mice and this trend has culminated in the rapid deterioration of the bitter-tasting capability in humans. Since T2R molecules play an important role in avoiding generally bitter toxic and harmful substances, substantial modification of the T2R gene repertoire is likely to reflect different responses to changes in the environment and to result from species-specific food preference during primate evolution.     

Negligible nuclear introgression despite complete mitochondrial capture between two species of chipmunks

The idea that species boundaries can be semipermeable to gene flow is now widely accepted but the evolutionary importance of introgressive hybridization remains unclear. Here we examine the genomic contribution of gene flow between two hybridizing chipmunk species, Tamias ruficaudus and T. amoenus. Previous studies have shown that ancient hybridization has resulted in complete fixation of introgressed T. ruficaudus mitochondrial DNA (mtDNA) in some populations of T. amoenus, but the extent of nuclear introgression is not known. We used targeted capture to sequence over 10,500 gene regions from multiple individuals of both species. We found that most of the nuclear genome is sorted between these species and that overall genealogical patterns do not show evidence for introgression. Our analysis rules out all but very minor levels of interspecific gene flow, indicating that introgressive hybridization has had little impact on the overall genetic composition of these species outside of the mitochondrial genome. Given that much of the evidence for introgression in animals has come from mtDNA, our results underscore that unraveling the importance introgressive hybridization during animal speciation will require a genome‐wide perspective that is still absent for many species.     

Pedigree models for complex human traits involving the mitochondrial genome.

Recent biochemical and molecular-genetic discoveries concerning variations in human mtDNA have suggested a role for mtDNA mutations in a number of human traits and disorders. Although the importance of these discoveries cannot be emphasized enough, the complex natures of mitochondrial biogenesis, mutant mtDNA phenotype expression, and the maternal inheritance pattern exhibited by mtDNA transmission make it difficult to develop models that can be used routinely in pedigree analyses to quantify and test hypotheses about the role of mtDNA in the expression of a trait. In the present paper, we describe complexities inherent in mitochondrial biogenesis and genetic transmission and show how these complexities can be incorporated into appropriate mathematical models. We offer a variety of likelihood-based models which account for the complexities discussed. The derivation of our models is meant to stimulate the construction of statistical tests for putative mtDNA contribution to a trait. Results of simulation studies which make use of the proposed models are described. The results of the simulation studies suggest that, although pedigree models of mtDNA effects can be reliable, success in mapping chromosomal determinants of a trait does not preclude the possibility that mtDNA determinants exists for the trait as well. Shortcomings inherent in the proposed models are described in an effort to expose areas in need of additional research.     

Humanin拮抗AD相关毒性作用的研究进展

阿尔采末病（Alzheimer＇s disease,AD）严重威胁着老年人的健康与生存质量,由于其发病原因复杂、机制不明,目前尚缺乏有效的防治措施。Humanin是近年来发现的能特异性抑制AD相关毒性的分泌性短肽,通过自身二聚化在细胞外发挥神经保护作用。Humanin通过直接或间接作用抑制β-淀粉样蛋白（AB）,以及家族性AD（FAD）基因如早老基因突变诱发的神经毒,而对AD不相关的毒性作用如凋亡诱导剂etoposide及Fas诱导的细胞死亡却不表现拮抗作用。因此,Humanin可通过特异性拮抗AD相关的神经毒而发挥重要的神经保护作用。