Molecular Exercise Physiology Mitochondrial biogenesis Presentation 3 Henning Wackerhage

At the end of this presentation you should be able to: Explain the endosymbiosis hypothesis. Describe stimuli that activate mitochondrial biogenesis. Explain the effect of an overexpression of PGC-1 in muscle. Explain how the expression of mitochondrial proteins encoded in nuclear and mitochondrial DNA is regulated. Learning outcomes Mitochondrial biogenesis is not an easy process to understand. You will need to spend considerable time on revision.

Mitochondrial biogenesis Part 1 Exercise and mitochondrial biogenesis

Mitochondrial DNA Krebs cycle Electron transport chain F 0 F 1 ATP synthase Pathways (e.g. b-oxidation) Transporters (e.g. ATP/ADP, FAs) Mitochondrial biogenesis Mitochondria are the power stations of our cells and the sites of oxidative phosphorylation. The enzymes for fat metabolism (- oxidation), the Krebs cycle, electron transport chain and finally the F 0 F 1 ATP synthase are located inside the mitochondria. Hopefully, you know a bit about oxidative phosphorylation from previous lectures. Have a close look at the parts of a mitochondrion in the figure below. All these parts need to be synthesized during mitochondrial biogenesis.

Task How do mitochondria synthesize ATP? Name at least two researchers that won a Nobel price on ATP synthesis by mitochondria.

Mitochondrial biogenesis in skeletal muscle Mitochondrial biogenesis can be stimulated in skeletal muscle: Skeletal muscle mitochondria proliferate (divide and increase in numbers) in response to exercise (Holloszy et al 1967). Chronic electrical stimulation of a muscle also increasesd mitochondrial biogenesis (Williams et al. 1987). Thyroid hormones increase the metabolic rate and mitochondrial enzyme levels (Tat et al. 1963). Mitochondrial biogenesis also occurs during development.

Chronic electrical stimulation activates mitochondrial biogenesis Control Salmons, Jarvis, Higginson, Manolopoulos, Woods, Wackerhage, unpublished data (2001) Increased mitochondrial density after chronic electrical stimulation In this study, an electrode was connected to a motor nerve innervating the hindlimbs of a rat. The hindlimb muscles were stimulated for several weeks. The stain is the nitro blue tetrazolium stain which stains a mitochondrial enzyme. The figure shows how chronic electrical stimulation increases the mitochondrial content of this muscle.

Saltin et al. (1976) Subjects endurance trained with one leg and rested the other. After the training period, the capillary density, mitochondrial content and peak oxygen uptake achieved when cycling with that leg were measured. The results showed that endurance training increased the mitochondrial content of the trained leg by  20% and also the peak oxygen uptake that was achieved when working with the trained leg only. Endurance training increases the mitochondrial content

Mitochondrial biogenesis Part 2 Origin of mitochondria

Serial endosymbiosis hypothesis Where do mitochondria come from? Lynn Margulis published a book in 1981 entitled “symbiosis in cell evolution”, where she stated the so-called “endosymbiosis hypothesis”. According to this hypothesis, cells with a pre-aerobic metabolism invaded anaerobic, prokaryotic host cells and formed a symbiosis. The most compelling evidence for the endosymbiotic hypothesis is that mitochondria have their own DNA. Less than 10 % of the proteins of a mitochondrion are encoded in the mitochondrial DNA. All other proteins are encoded in the nuclear DNA. Prof Lynn Margulis

Eukaryote with aerobic metabolism Ancestral host cell (anaerobic) Serial endosymbiosis hypothesis Ancestral prokaryote (pre-aerobic metabolism)

Two DNAs encode mitochondrial proteins Mitochondrial DNA Nuclear DNA encoding mitochondrial proteins Because there are two sources of DNA that encode mitochondrial biogenesis, a regulatory system is needed to regulate the expression of these proteins, and their transport and assembly during mitochondrial biogenesis.

Mitochondrial biogenesis Part 4 Mitochondrial genetics

Sequencing of mitochondrial DNA All genes that encode mitochondrial proteins need to be expressed during mitochondrial biogenesis. Some of these genes are encoded in mitochondrial DNA and others in the “normal” DNA inside the nucleus. Researchers at the University of Cambridge have published the DNA sequence of the human mitochondrial DNA in 1981, which can be considered as the start of the human genome project: Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith AJ, Staden R, Young IG. Sequence and organization of the human mitochondrial genome. Nature. 290: , This paper showed that a DNA sequence that contained base pairs could be sequenced with the DNA sequencing techniques developed by Fred Sanger and others.

Anderson et al. (1981), Clayton (1991) Mitochondrial DNA What is the structure of mitochondrial DNA? Mitochondrial DNA contains base pairs and it is transcribed as a single transcript that encodes 13 proteins (see figure). The mitochondrial DNA is economical: there are very few non-coding sequences. Mitochondrial DNA encodes only 13 proteins which is less than 10 % of all the mitochondrial proteins. All other proteins are encoded in the nuclear DNA. Transcription factors that regulate the transcription and replication (doubling) of DNA bind to the displacement loop (D-loop, see top of figure).

Task a) Identify the names of the genes that are encoded in mitochondrial DNA. Name at least five other proteins that are needed for the production of mitochondria. b) How is mitochondrial DNA inherited. Find out.

Complex Mitochondrial Nuclearencoded proteins I7>25 II04 III110 IV 310 F 0 F 1 ATP synthase 211 Poyton and McEwan (1996) The respiratory chain in mitochondria consists of four protein complexes plus F 0 F 1 ATP synthase which is synthesizing ATP. All complexes are assembled from several proteins. The table shows that some of these proteins are encoded in nuclear DNA and others in mitochondrial DNA. Mitochondrial DNA

Mitochondrial biogenesis Part 3 Factors and pathways involved

Task Assume you would have to design a regulatory mechanism by which exercise activates mitochonrial biogenesis. What would be a good exercise signal in muscle? How would you activate mitochondrial genes encoded in nuclear DNA? How would you activate mitochondrial genes encoded in mitochondrial DNA? What other steps and reactions are needed?

Mitochondrial biogenesis requires co-ordination of the synthesis of mitochondrial proteins and other molecules and the assembly of these molecules into a new mitochondrion. Researchers have identified two classes of transcription factors that are involved in this process: a) Transcription factors that regulate the transcription and replication (DNA copying) of mitochondrial DNA (mtDNA); b) Transcription factors that regulate the transcription of mitochondrial genes encoded in the nuclear DNA. Regulation of mitochondrial biogenesis Transcription factor regulating mitochondrial genes in nuclear DNA Nucleus Mitochondria

Several transcription factors have been discovered by researchers. Leading in the field are Richard Scarpulla, who has discovered the nuclear respiratory factors (NRF-1, NFRF-2) and Pere Puigserver who discovered the transcriptional co-factor PPAR coactivator (PGC-1). This co-factor is not a transcription factor itself but binds to transcription factors. These transcription and co-factors regulate the expression of mitochondrial genes located in the nucleus. Richard C ScarpullaPere Puigserver Transcription factors acting on nuclear DNA

The nuclear respiratory factor (NRF-1) was discovered by Evans and Scarpulla (1989, 1990). Subsequent studies showed that NRF- 1 had transcription factor binding sites in genes that encoded proteins of the respiratory chain, F 0 F 1 ATP synthase, heme biosynthesis and protein import into mitochondria. All these proteins are located in the nuclear DNA. Many of these genes also have binding sites for NRF-2. Puigserver et al. (1998) then discovered the transcriptional co- activator PGC-1. Both NRF-1, PGC-1 and other transcription and co-factors bind together and regulate the expression of mitochondrial genes that are encoded in nuclear DNA. NRF PGC-1 Expression of mitochondrial genes

Task PGC-1 is a transcriptional co-activator. What is the difference between a transcriptional co-activator and a transcription factor? How do they work?

PGC-1 and mitochondrial biogenesis PGC-1 AMPK CamK p38 Endurance exercise The story so far: AMP-activated protein kinase (AMPK; Terada et al. 2003) and Calcium/calmodulin-activated kinase (CamK IV; Wu et al. 2003) have been shown to stimulate PGC-1 expression. The action of p38 MAPK leads to an increased phosphorylation of PGC-1 (Puigserver et al. 2003).

PGC-1 and mitochondrial biogenesis ? PGC-1 NRF mtTFA AMPK CamK p38 Mitochondrial biogenesis Endurance exercise The more detailed story: PGC-1 An increased PGC-1 concentration then expression of NRF-1, NRF-2 and mtTFA, i.e. transcription factors that regulate mitochondrial genes encoded in nuclear and mitochondrial DNA. In addition, PGC- 1 binds to NRF-1 (Wu et al. 1999). Thus, PGC-1 appears to be the master regulator of mitochondrial biogenesis.

PGC-1 is the master regulator of mitochondrial biogenesis Lin et al. (2002) generated transgenic mice that overexpressed PGC-1 in their muscles. In the figures, WT refers to the wildtype (normal mice) and TG to the transgenic mice. The transgenic muscles have more cytochrome c, an enzyme found in mitochondria. These data show that PGC-1 can induce mitochondrial biogenesis in vivo. WT TG

PGC-1 is the master regulator of mitochondrial biogenesis and also regulates other “slow” genes Overexpression of PGC-1 did not only affect mitochondrial biogenesis. The transgenic muscles appear red due to the high myoglobin content (see also the Western blot bottom left. The mitochondrial content is increased as expected (cytochrome C is a marker) and surprisingly also motor proteins such as slow troponin (TnI) and slow myosin are upregulated (Lin et al. 2002).

AMPK induces PGC-1 and mitochondrial biogenesis PGC-1 is also induced by CamK IV (see next slide) and phosphorylated by p38 MAPK. All three, AMPK, CamK and p38 have been shown to be activated by exercise, induce or activate PGC-1 and induce mitochondrial biogenesis. The following figure shows that swimming and AICAR, an AMPK activator, increase PGC-1 (Terada et al. 2002).

AMPK was named here at Dundee AMPK was named by Prof. Grahame Hardie, who know works at the Wellcome Trust Biocentre at the University of Dundee. AMPK regulates the adaptation to exercise and is a treatment target for type 2 diabetes mellitus and obesity. Grahame Hardie AMPK AMP ATP, PCr AMPK regulation

Wu et al. (2002) generated transgenic mice that overexpressed calmodulin- dependent kinase IV (CaMk IV) in their muscles. In the figures, WT refers to the wildtype, the normal mouse and TG to the transgenic mouse. The transgenic mice had more mitochondria in their muscles (round objects in the section). In addition they measure the cytochrome B gene DNA which is a marker for the mitochondrial DNA content. CaMK IV induces PGC-1 and mitochondrial biogenesis

Transcription factors acting on mitochondrial DNA OK, nuclear genes encoding mitochondrial proteins are regulated by the co-factor PGC-1 and transcription factors such as NRF-1. However, the expression of these genes is not enough for mitochondrial biogenesis. The question is: (1) Which transcription factors stimulate the transcription of mitochondrial DNA and (2) the replication of mitochondrial DNA? The latter is important because the new mitochondria need their own mitochondrial DNA. The following slide gives an answer to this question. mtDNA (1) (2) Endurance exercise ?

Transcription factors acting on mitochondrial DNA NRF-1 (together with PGC-1) was also shown to induce the mitochondrial transcription factor A (mtTFA; also known as Tfam, TCF6). mtTFA is imported into mitochondria and binds to the D-loop of the mitochondrial DNA. mtTFA binding to mitochondrial DNA induces the replication and transcription of mitochondrial DNA. Knockout mice that do not synthesize mtTFA die in the uterus because they do not synthesize mitochondria. The sequence of events is shown below. NRF-1 PGC-1 (2) mtTFA transcription (3) mtTFA translation (4) mtTFA is imported to the mitochondrion and binds to mitochondrial DNA which is then replicated and transcribed. (1) Endurance exercise mtDNA mtTFA

How mitochondrial biogenesis works Putting it all together (see schematical figure on the next slide): Exercise activates the AMPK, CamK and p38 signal transduction pathways, among other. A key consequence is the increased expression and phosphorylation of the transcriptional co-factor PGC-1. The exact mechanisms are largely unknown. PGC-1 with transcription factors such as MEF2 and PPAR is increasing the expression of mitochondrial transcription factors such as NRF-1. PGC-1 now together with transcription factors such as NRF-1 is causing an increased transcription of a) mitochondrial genes encoded in nuclear DNA and b) the mitochondrial transcription factor mtTFA. mtTFA is causing the transcription of the genes encoded in mitochondrial DNA and replication of mitochondrial DNA. All proteins encoded in nuclear and mitochondrial DNA together with all other parts are assembled as a mitochondrion.

Task Stop here. Try to draw a diagram from the information given on the previous slide.

Nucleus Skeletal muscle fibre PGC-1 expression AMPK AMP, PCr PPAR PGC-1 MEF2 NRF- expression CaMK [Ca] i  Mitochondrial protein encoded in nuclear DNA Mitochondrial protein encoded in mitochondrial DNA mtTFA and mtTFB Replication and transcription Exercise-induced mitochondrial biogenesis Exercise ? Mitochondrion Protein import and assembly Mitochondrial biogenesis NRF PGC-1 Expression of mtTFs and proteins p38

The End