Effect of alternative metabolic fuels as a potential ALS therapy in mice and humans

Csilla Ari, Craig R. Goldhagen, Angela Poff, Heather Held, Carol Landon, Nicholas Mavromates Dominic D. D’Agostino

Department of Molecular Pharmacology and Physiology, Hyperbaric Biomedical Research Laboratory, Morsani College of Medicine, University of South Florida, Tampa FL

Amyotrophic Lateral Sclerosis (ALS) more commonly known as Lou Gehrig’s disease, is a neurodegenerative disorder of motor neurons causing progressive muscle weakness, paralysis, and finally death from respiratory failure within 3 to 5 years after diagnosis. There is currently no cure or effective treatment for ALS. Motor neuron death results from impaired energy metabolism, which is linked pathophysiologically to mitochondrial dysfunction, oxidative stress, glutamate excitotoxicity. In normal conditions the human body will use glucose as the main metabolic fuel; however, neurodegenerative disorders are known to have impaired glucose transport and metabolism. These neurodegenerative cells require an alternative metabolic substrates and cofactors to preserve cellular function, resting membrane potential and prevent glutamate toxicity. Metabolic therapy that supplies alternative energy substrates and cofactors may enhance mitochondrial function and represent a viable therapeutic approach for ALS. In this study we hypothesized that alternative fuels in the form of ketone bodies, specifically arginine-alpha-ketoglutarate (AAKG) and the ketogenic diet (KD; 80% fat), would increase motor function, slow the progression of the disease and extend survival in a mouse model of ALS (SOD1G93A mice).

Animal Model• Transgenic SOD1G93A mice (Jackson Laboratory) are currently the only

ALS model exhibiting all of the pathological characteristics of ALS

Deanna Protocol (DP) Metabolic Therapy for ALSComposition of DP in Foof• AAKG (Arginine Alpha-ketoglutarate) Primaforce, 10%• GABA (Gamma-aminobutyric acid ) Primaforce, 1%• CoEQ10 ( Ubiquinol)Tishcon, 0.1%• Caprylic acid (MCT oil) Life enhancement, 1%

Dietary Groups • Control (SD) – Standard Diet• SDDP – Standard Diet combined with Deanna Protocol• KD – Ketogenic Diet • KDDP – Ketogenic Diet combined with the Deanna Protocol

Standard Diet: Low Fat and High CarbohydratesKetogenic Diet: High Fat and Low Carbohydrates

Experiments

• Motor Function Tests (rotarod, grip test, hanging wire test)

• Neurological Score (0 to 4) : 0 = no pathology

• Survival (defined criteria)

Experimental Design

Background Summary

• KD group had greatly improved motor function during the first stages of disease onset (weeks 5 and 6) when compared with the SD, however, the KD did not extend survival time.

• During the later stages of the disease (weeks 7 and 8) the SDDP group had significantly better motor function.

• Neurological Score in the SDDP group was improved toward late stages of disease. KD provided no significant benefit in Neurological Score alone or when combined with the Deanna Protocol.

• Our data shows increased survival in SDDP treatment group

ReferencesZhao W, Varghese M, Vempati P, Dzhun A, Cheng A, Wang J, Lange D, Bilski A, Faravelli I, Pasinetti GM. Caprylic triglyceride as a novel therapeutic approach to effectively improve the performance and attenuate the symptoms due to the motor neuron loss in ALS disease. PLoS One. 2012;7(11):e49191Zhao Z, Lange DJ, Voustianiouk A, MacGrogan D, Ho L, Suh J, Humala N, Thiyagarajan M, Wang J, Pasinetti GM. A ketogenic diet as a potential novel therapeutic intervention in amyotrophic lateral sclerosis. BMC Neuroscience. 2006 Apr 3;7:29

AcknowledgementsThis work was supported by Winning the Fight against Neurodegenerative Diseases (WFND), a 501(c)3 non-profit dedicated to raising awareness about neurodegenerative diseases (NDs), as well as seeking and funding new treatments for NDs.

For more information see: www.winningthefight.net

Contact: Dominic D’Agostino: ddagosti@health.usf.edu Csilla Ari: csari2000@yahoo.com

Results

Motor Function Neurological Score

Survival

KD and SD+DP Enhanced Motor FunctionFig2 A) KD group had significantly better result on accelerating rotarod during week 11, 15 and 16 (p=0.029, p=0.004, p=0.036, respectively) when compared to the control group, but during week 17 and 18 (p=0.039, p=0.046, respectively) only the SD+DP group performed better. B) Grip test showed better motor performance in KD group at week 15 (p=0.015), while strength was significantly better at week 17 and 18 in SD+DP group (p=0.039, p=0.046), compared to control.C) Hanging Wire Test test showed better motor performance in KD group on week 16 (p=0.036), while performance was significantly better at week 16 and 17 in SD+DP group (p=0.028, p=0.037), when compared to SD group. All data are mean ± SEM.

Results

Figure 3 A) Neurological Score was significantly improved in SD+DP group between day 118 and 136, suggesting delayed disease progression in SD+DP group (p˂0.05). All data are mean ± SEM.

Figure 3 B) Kaplan-Meier survival plot of study groups. Animals receiving SD+DP showed significantly longer survival compared to control animals (p=0.001).

Score of 0 Hind legs are stretched fully when hanging by tailScore of 1 Hind legs are pulled back when hanging by tailScore of 1.5 Abnormal gaitScore of 2 Toes curled under when walkingScore of 2.5 Use of both hind legs while walking, but with difficultyScore of 3 Pulling back legs, but still able to move them bothScore of 3.5 One of the hind legs paralyzed, with use of the otherScore of 4 Completely paralyzed or dead

Table 1. Neurological score: Table contains criteria for each neurological score

Figure 1: Experimental Time Line for the study

Grip Test

Hanging Wire Test

Rotarod performanceA

B

C

In conclusion, targeting energy metabolism with a metabolic therapy produces a beneficial effect in ALS mice and is a feasible strategy that may prolong survival and quality of life of ALS patients.