Janne M. Toivonen

Mechanisms of Life Span Extension by Rapamycin in the Fruit Fly Drosophila melanogaster

Summary The target of rapamycin (TOR) pathway is a major nutrient-sensing pathway that, when genetically downregulated, increases life span in evolutionarily diverse organisms including mammals. The central component of this pathway, TOR kinase, is the target of the inhibitory drug rapamycin, a highly specific and well-described drug approved for human use. We show here that feeding rapamycin to adult Drosophila produces the life span extension seen in some TOR mutants. Increase in life span by rapamycin was associated with increased resistance to both starvation and paraquat. Analysis of the underlying mechanisms revealed that rapamycin increased longevity specifically through the TORC1 branch of the TOR pathway, through alterations to both autophagy and translation. Rapamycin could increase life span of weak insulin/Igf signaling (IIS) pathway mutants and of flies with life span maximized by dietary restriction, indicating additional mechanisms.

In Vivo Functional Analysis of the Human Mitochondrial DNA Polymerase POLG Expressed in Cultured Human Cells

The human gene POLG encodes the catalytic subunit of mitochondrial DNA polymerase, but its precise roles in mtDNA metabolism in vivo have not hitherto been documented. By expressing POLG fusion proteins in cultured human cells, we show that the enzyme is targeted to mitochondria, where the Myc epitope-tagged POLG is catalytically active as a DNA polymerase. Long-term culture of cells expressing wild-type POLG-myc revealed no alterations in mitochondrial function. Expression of POLG-myc mutants created dominant phenotypes demonstrating important roles for the protein in mtDNA maintenance and integrity. The D198A amino acid replacement abolished detectable 3′-5′ (proofreading) exonuclease activity and led to the accumulation of a significant load (1:1700) of mtDNA point mutations during 3 months of continuous culture. Further culture resulted in the selection of cells with an inactivated mutator polymerase, and a reduced mutation load in mtDNA. Transient expression of POLG-myc variants D890N or D1135A inhibited endogenous mitochondrial DNA polymerase activity and caused mtDNA depletion. Deletion of the POLG CAG repeat did not affect enzymatic properties, but modestly up-regulated expression. These findings demonstrate that POLG exonuclease and polymerase functions are essential for faithful mtDNA maintenance in vivo, and indicate the importance of key residues for these activities.

Endocrine regulation of aging and reproduction in Drosophila.

Hormonal signals can modulate lifespan and reproductive capacity across the animal kingdom. The use of model organisms such as worms, flies and mice has been fundamentally important for aging research in the discovery of genetic alterations that can extend healthy lifespan. The effects of mutations in the insulin and insulin-like growth factor-like signaling (IIS) pathways are evolutionarily conserved in that they can increase lifespan in all three animal models. Additionally, steroids and other lipophilic signaling molecules modulate lifespan in diverse organisms. Here we shall review how major hormonal pathways in the fruit fly Drosophila melanogaster interact to influence reproductive capacity and aging.

No Influence of Indy on Lifespan in Drosophila after Correction for Genetic and Cytoplasmic Background Effects

To investigate whether alterations in mitochondrial metabolism affect longevity in Drosophila melanogaster, we studied lifespan in various single gene mutants, using inbred and outbred genetic backgrounds. As positive controls we included the two most intensively studied mutants of Indy, which encodes a Drosophila Krebs cycle intermediate transporter. It has been reported that flies heterozygous for these Indy mutations, which lie outside the coding region, show almost a doubling of lifespan. We report that only one of the two mutants lowers mRNA levels, implying that the lifespan extension observed is not attributable to the Indy mutations themselves. Moreover, neither Indy mutation extended lifespan in female flies in any genetic background tested. In the original genetic background, only the Indy mutation associated with altered RNA expression extended lifespan in male flies. However, this effect was abolished by backcrossing into standard outbred genetic backgrounds, and was associated with an unidentified locus on the X chromosome. The original Indy line with long-lived males is infected by the cytoplasmic symbiont Wolbachia, and the longevity of Indy males disappeared after tetracycline clearance of this endosymbiont. These findings underscore the critical importance of standardisation of genetic background and of cytoplasm in genetic studies of lifespan, and show that the lifespan extension previously claimed for Indy mutants was entirely attributable to confounding variation from these two sources. In addition, we saw no effects on lifespan of expression knockdown of the Indy orthologues nac-2 and nac-3 in the nematode Caenorhabditis elegans.

MicroRNA-206: A Potential Circulating Biomarker Candidate for Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a lethal motor neuron disease that progressively debilitates neuronal cells that control voluntary muscle activity. Biomarkers are urgently needed to facilitate ALS diagnosis and prognosis, and as indicators of therapeutic response in clinical trials. microRNAs (miRNAs), small posttranscriptional modifiers of gene expression, are frequently altered in disease conditions. Besides their important regulatory role in variety of biological processes, miRNAs can also be released into the circulation by pathologically affected tissues and display remarkable stability in body fluids. In a mouse model of ALS that expresses mutated human superoxide dismutase 1 (SOD1-G93A) skeletal muscle is one of the tissues affected early by mutant SOD1 toxicity. To find biomarkers for ALS, we studied miRNA alterations from skeletal muscle and plasma of SOD1-G93A mice, and subsequently tested the levels of the affected miRNAs in the serum from human ALS patients. Fast-twitch and slow-twitch muscles from symptomatic SOD1-G93A mice (age 90 days) and their control littermates were first studied using miRNA microarrays and then evaluated with quantitative PCR from five age groups from neonatal to the terminal disease stage (10–120 days). Among those miRNA changed in various age/gender/muscle groups (miR-206, -1, -133a, -133b, -145, -21, -24), miR-206 was the only one consistently altered during the course of the disease pathology. In both sexes, mature miR-206 was increased in fast-twitch muscles preferably affected in the SOD1-G93A model, with highest expression towards the most severely affected animals. Importantly, miR-206 was also increased in the circulation of symptomatic animals and in a group of 12 definite ALS patients tested. We conclude that miR-206 is elevated in the circulation of symptomatic SOD1-G93A mice and possibly in human ALS patients. Although larger scale studies on ALS patients are warranted, miR-206 is a promising candidate biomarker for this motor neuron disease.

Altered Expression of Myogenic Regulatory Factors in the Mouse Model of Amyotrophic Lateral Sclerosis

Background: In the superoxide dismutase 1 (SOD1)-G93A mouse model of amyotrophic lateral sclerosis (ALS), skeletal muscle is a key target of mutant SOD1 toxicity. However, the expression of factors that control the regenerative potential of the muscle is unknown in this model. Objective: To characterize the expression of satellite cell marker Pax7 and myogenic regulatory factors (MRF) in skeletal muscle of SOD1-G93A mice at different stages of the disease. Methods: The expressions of Pax7, Myod1, Myf5 and myogenin (Myog) were determined by quantitative real-time PCR and by Western blotting from the grouped gastrocnemius, quadriceps and soleus muscles of SOD1-G93A mice at presymptomatic, symptomatic and terminal stages of the disease, and from surgically denervated wild-type gastrocnemius muscles. Results:Pax7 mRNA and MYF5 protein were upregulated in presymptomatic mice, coinciding with increased muscle damage marker Rrad and chemokine Ccl5. All MRF transcripts and most proteins (excluding MYOG) were increased, starting from 3 months of age, simultaneously with increased expression of denervation marker Chrna1. However, in the terminal stage, no protein increase was evident for Pax7 or any of the MRF despite the increased mRNA levels. The transcripts for chemokine Ccl2 and chemokine receptor Cxcr4 were increased starting from the onset of symptoms. Conclusions: The characterization of Pax7 and MRF in SOD1-G93A mice reveals a progressive induction of the myogenic program at the RNA level, but a blunted protein level response at late stages of the disease. Altered posttranscriptional and posttranslational mechanisms likely to operate, as well as the potential role of chemokine signaling in mutant SOD1 muscle, are discussed.

Tetanus toxin C-fragment: the courier and the cure?

In many neurological disorders strategies for a specific delivery of a biological activity from the periphery to the central nervous system (CNS) remains a considerable challenge for successful therapy. Reporter assays have established that the non-toxic C-fragment of tetanus toxin (TTC), provided either as protein or encoded by non-viral naked DNA plasmid, binds pre-synaptic motor neuron terminals and can facilitate the retrograde axonal transport of desired therapeutic molecules to the CNS. Alleviated symptoms in animal models of neurological diseases upon delivery of therapeutic molecules offer a hopeful prospect for TTC therapy. This review focuses on what has been learned on TTC-mediated neuronal targeting, and discusses the recent discovery that, instead of being merely a carrier molecule, TTC itself may well harbor neuroprotective properties.

Sex, fiber‐type, and age dependent in vitro proliferation of mouse muscle satellite cells

During postnatal growth and after muscle injury, satellite cells proliferate and differentiate into myotubes to form and repair musculature. Comparison of studies on satellite cell proliferation and differentiation characteristics is confounded by the heterogeneity of the experimental conditions used. To examine the influence of sex, age, and fiber‐type origin on in vitro properties of satellite cells derived from postnatal muscles, fast extensor digitorum longus (EDL) and slow soleus (SOL) muscles were extracted from male and female mice of 1 week to 3 months of age. Myoblast proliferation and myogenic regulatory factor (MRF) expression was measured from cultures of freshly isolated satellite cells. Higher proliferation rate and elevated Myod1 expression was found in male EDL and SOL derived cells compared with females at age of 40, 60, and 120 days, whereas inverse tendency for cell proliferation was apparent in EDL of juvenile (7‐day‐old) pups. Myogenin and Mrf4 transcripts were generally elevated in males of 40 and 60 days of age and in female EDL of juveniles. However, these differentiation markers did not significantly correlate with proliferation rate at all ages. Pax7, whose overexpression can block myogenesis, was up‐regulated especially in 40‐day‐old females where MRF expression was low. These results indicate that gender, postnatal age, and muscle fiber origin affect proliferation and muscle transcription factor expression in vitro. The results also support the view that satellite cells originating from slow and fast muscles are intrinsically different and warrant further studies on the effect of cell origin for therapeutic approaches. J. Cell. Biochem. 112: 2825–2836, 2011.

Modelling in Escherichia coli of mutations in mitoribosomal protein S12: novel mutant phenotypes of rpsL

The rpsL gene of Escherichia coli encodes the highly conserved rps12 protein of the ribosomal accuracy centre. We have used the E. coli gene to model the phenotypic effects of specific substitutions found in the mitochondrial gene for rps12. Variants created by in vitro mutagenesis were tested in two different plasmid vector systems, in both streptomycin‐sensitive and streptomycin‐resistant hosts. A substitution with respect to eubacterial rps12 (K87→Q), found in all metazoan and fungal mitochondrial orthologues thus far studied, is associated with low‐level resistance to streptomycin and a modest (15%) drop in translational elongation rate, but without significant effects on translational accuracy. An amino‐acid replacement at a highly conserved leucine residue (L56→H), associated with the phenotypes of sensitivity to mechanical vibration and hemizygous female lethality in Drosophila, creates a functionally inactive but structurally stable protein that is not assembled into ribosomes. The presence in the cell of the mutant, but not wild‐type, rpsL greatly downregulates the level of a prominent polypeptide of ≈ 50 kDa. These results indicate novel structure–function relationships in rps12 genes affecting translational function, ribosome assembly and drug sensitivity, and indicate a novel regulatory pathway that may influence ribosome biogenesis.

Altered in vitro proliferation of mouse SOD1-G93A skeletal muscle satellite cells.

Background: Amyotrophic lateral sclerosis (ALS) is the most common adult-onset neurodegenerative disease characterized by ascending muscle weakness, atrophy and paralysis. Early muscle abnormalities that precede motor neuron loss in ALS may destabilize neuromuscular junctions, and we have previously demonstrated alterations in myogenic regulatory factor (MRF) expression in vivo and in the activation of myofiber-associated skeletal muscle satellite cells (SMSCs) in the mouse model of ALS (SOD1-G93A). Methods: To elucidate niche dependence versus cell-autonomous mutant SOD1 (mSOD1) toxicity in this model, we measured in vitro proliferation potential and MRF and cyclin gene expression in SMSC cultures derived from fast-twitch extensor digitorum longus and slow-twitch soleus muscles of SOD1-G93A mice. Results: SMSCs from early presymptomatic (p40) to terminal, semi-paralytic (p120) SOD1-G93A mice demonstrated generally lower proliferation potential compared with age-matched controls. However, induced proliferation was observed in surgically denervated wild-type animals and SOD1-G93A animals at p90, when critical denervation arises. SMSCs from fast and slow muscles were similarly affected by mSOD1 expression. Lowered proliferation rate was generally corroborated with decreased relative MRF expression levels, although this was most prominent in early age and was modulated by muscle type origin. Cyclins controlling cell proliferation did not show modifications in their mRNA levels; however, the expression of cyclin-dependent kinase inhibitor 1A (Cdkn1a), which is known to promote myoblast differentiation, was decreased in SOD1-G93A cultures. Conclusions: Our data suggest that the function of SMSCs is impaired in SOD1-G93A satellite cells from the earliest stages of the disease when no critical motor neuron loss has been described.