NAD+-Dependent Activation of Sirt1 Corrects the Phenotype in

Results #1

(continued)

Key:

Solid Black: WT (a)

Dashed Black: PARP1 (-/-) (b)

Solid Red: Sco2 (c)

Dashed Red: double mutant (d)

Results #3

PARP1 Inhibitors Induce Mitochondrial Function in Skeletal Muscle and Brain

Subjects: KOKI mice & WT

Results #2 (Contd)

• Administered an inhibitor (MRLB) of PARP1
• Weekly treadmill performance increased in Sco2 mice, no change in WT seen
• Sirt1 activated in both groups
• Western blot analysis showed increased content of mitochondrial respitory subunits in Sco2 mouse
• In contrast to the NR treatment, the inhibitor determined significant increase of respiratory chain activities in the brain.

Results #2

NR Increases Motor Performance and Induces OXPHOS- Related Gene Expression in Sco2 Skeletal Muscle

Key:

Solid Black: WT

Dashed Black: WT+NR

Solid Red: Sco2

Dashed Red: Sco2 + NR

Subjects: WT & KOKI (Sco2)

Results #3(contd)

• Increased mitochondrial function and levels of motor endurance (Sco2)
• Increased mRNA levels (Sco2)
• Increased protein levels (Sco2)
• Increased levels of mitochondrial respiratory chain activities (Sco2)
• NAD+/NADH ratio increases (WT and Sco2)
• Transcripts related to mtUPR were increased while those unrelated were unchanged in Sco2 mice

*Mitochondrial Unfolded Protein Response (mtUPR):

a stress response protective mechanism which can improve

mitochondrial function.

Key:

Solid Black: WT

Dashed Black: WT+MRLB

Solid Red: Sco2

Dashed Red: Sco2 + MRLB

Amarachi Uwaga

Edeline Sanchez

Ran Fontenelle

Stephanie Igbinovia

Methods

• In Vivo
• Western Blot Analysis
• PCR
• Locomotor

Results #1

Ablation of PARP1 Improved Motor Performance in Sco2 Mouse

Subjects Used: Sco2 mice, PARP1(-/-) mice, WT mice & Sco2/PARP1 mice

Conclusion

• Double mutants showed increased levels of NAD+
• Reduced fasting blood glucose levels, body weight, epididymal white adipose tissue compared to Sco2 mice
• Sco2 mice showed reduced motor performance compared to WT litter mates
• Double mutants performed as well as WT and PARP litter mates
• COX was increased in skeletal muscle of the double mutants showed in Figure C
• Activities of CIII and CIV were also increased in the brains of the double mutants

• Removal of the PARP1 gene, supplementation of NR or administration of Parp inhibitors can expand the NAD+ pool and active sirtuins (Sirt1), a master regulator of mitochondria homeostasis
• Regulating the mitochondria homeostasis produce effects that protect mice from high fat induced- metabolic disease.
• These treatments used correct both the biochemical and clinical phenotype of the Sco2 mouse

Study Subjects

• Sco2 mouse- defect
• WT- wild type, control
• Parp1(-/-)- removed from mice
• Sco2/Parp1- double mutant

Motor performance of the four subjects, (4 animals per group), was monitored weekly by a standard treadmill test for 4 weeks starting at 2 months of age.

Study Design

*Sco2 gene: Involved in the formation of cytochrome C oxidase, an enzyme in the electron transport chain

*Parp1 - NAD+ inhibitor

Experimental study

Controlled

Strengths:

Causation- allows inferences to be made about the relationship between the IV and DV

Control-allows for the ruling out of alternative explanations due to external variables

Variability- reduces variability within treatment conditions, making it easier to detect treatment outcomes

Weaknesses:

Artifical enviornment- low realism

Low Validity- diffucult to generalize to other subjects

Discussion

Hypothesis

Because Sirt1 directly targets the mitochondrial biogenesis pathways, thereby boosting mitochondrial function, and there is a direct relationship between Sirt1 and NAD+, the hypothesis is that NAD+ acts as a metabolic sensor.

*Metabolic sensors: Helps cell improvement

Study Objective

Discussion (contd)

• This research project has various practical uses and medical benefits.
• Prolonged NR supplementation up to 6 months induced mitochondrial biogenesis in the brain and improved cognitive dysfunction of an Alzheimer diseased mouse
• NR as a natural vitamin that is administered as a dietary supplement benefits reduced NAD+/NADH ratio, or aging
• PARP1 ablation or inhibition has been recorded to rescue models of liver cirrhosis by correcting the associated mitochondrial impairment.
• PARP1 inhibitors are currently under clinical trial as anticancer molecules

This research, using mice as models, show that with the supplementation of a natural NAD+ precursor, nicotinamide riboside, OR with the reduction of NAD+ consumption by inhibitors- poly (ADP-ribose) polymerases, there is an improvement in the disease.

The same theaureptic method used on the mice with the disease has potential to also treat humans with mitochondrial disorders.

• Because of the shorter time frame (4 weeks) compared to other experiments that last 12 weeks, time-dependent activation of different mitochondrial pathways were not able to be observed.
• Shorter time period did not allow for observations to be seen in the WT littermates.
• Limited number of subjects.

Background Info

• Mitochondrial diseases
• There are currently no effective therapies but research is continuously being done to increase the overall activity of the mitochondria and respiratory chain.
• Cytochrome C Oxidase Deficiency is the mitochondrial disease affecting the muscles used for movement (skeletal muscles) such as the heart, brain, or the liver.
• The gene mutations that cause cytochrome c oxidase deficiency affect an enzyme complex called cytochrome c oxidase, which is responsible for one of the final steps in oxidative phosphorylation.

References

How can I first understand the

title before analysis?

http://ghr.nlm.nih.gov/condition/cytochrome-c-oxidase-deficiency

http://highered.mcgraw-hill.com/sites/9834092339/student_view0/chapter7/how_the_nad__works.html

http://health.howstuffworks.com/wellness/aging/anti-aging-tips/anti-aging-gene1.htm

http://www.cell.com/abstract/S0092-8674(07)00682-4

• NAD+ - Nicotinamide Adenine Dinucleotide.
• NAD+ is an oxidizing agent
• Watch Video: -------------------------------------------->
• NAD+ activates the SIRT1, a protein also known as a dependent deacetylase Sirtuin 1.that is encoded by the Sirtuin 1 gene.
• It is an enzyme that deacetylases proteins
• Sirt1 activity is directly regulated by the availability of NAD+ and as NAD+ boosts Sirt1 activity

THANK YOU!

Any questions?

NAD+-Dependent Activation of Sirt1 Corrects the Phenotype in a Mouse Model of Mitochondrial Disease