Presentation on theme: "Glutamic-Oxaloacetic Transaminase Combined with Metabolic Therapy in a Mouse Model of Amyotrophic Lateral Sclerosis CQ Rogers, M Ramirez, CS Landon, JM."— Presentation transcript:
1 Glutamic-Oxaloacetic Transaminase Combined with Metabolic Therapy in a Mouse Model of Amyotrophic Lateral SclerosisCQ Rogers, M Ramirez, CS Landon, JM DeBlasi, AP Koutnik, and DP D’Agostino Department of Molecular Pharmacology and Physiology, Laboratory of Nutritional and Metabolic Medicine University of South Florida Health Sciences Center, Tampa FL
2 BACKGROUNDAmyotrophic lateral sclerosis (ALS) also known as Lou Gehrig's Disease is a progressive disease of neuronal degeneration in the spinal cord, brainstem and motor cortex resulting in impaired motor function and eventual demise. There is currently no widely accepted, effective treatment for this condition.Most cases of ALS (90%–95%) have no known cause and are termed sporadic ALS (sALS). In the remaining patients there is a history of disease in the family and so is known as familial ALS (fALS). Of these cases, 20%–25% have been mapped to the SOD1 (superoxide dismutase) gene on chromosome 21 where over 100 individual mutations have been identified. The mutation appears to cause an increase in the activity of the SOD1 protein which is involved in neutralizing oxidative damage.Lefty Lou Gehrig, the Iron Horse (c. 1934)
3 Glutamate + Oxaloacetate ↔ Alpha-Ketoglutarate + Aspartate INTRODUCTIONGlutamate (Glu) excitotoxicity is a process in which excessive stimulation of neurons can lead to their damage and death. The drug riluzole (Rilutek) is the only medication approved by the FDA for ALS. The drug appears to slow the disease's progression in some people, perhaps by reducing levels of Glu that is often present in higher levels in people with ALS.Oxaloacetate (OX) is an anaplerotic molecule and initiation point for the activation of Kreb’s Cycle. Previous work has demonstrated the ability of exogenous OX to reduce Glu levels.Glutamic-oxaloacetic transaminase (GOT) is an endogenous enzyme which catalyzes this reaction.Glutamate + Oxaloacetate ↔ Alpha-Ketoglutarate + AspartateGOT with OX has been shown to increase the removal of Glu from the blood and the CNS, thereby reducing excitotoxicity and improving neuronal health.
4 METHODSA novel metabolic therapy known as the Deanna Protocol (DP™ Plan) has been used effectively in ALS patients and mice. It consists of dietary supplements (arginine, alpha-ketoglutarate, medium-chain triglyceride oil, phenibut and coenzyme Q-10) to support mitochondria and the Kreb’s Cycle.Here we investigate the DP (DP™ Plan) alone or in conjunction with GOT and OX as a potential therapy.
5 Dosing / Bleed Schedule Preliminary Experiment: GOT 48 hr Pharmacokinetic StudyD'Agostino PK studyDosing / Bleed ScheduleMouse #DrugIP injectionBS 5 minBS 30 minBS 60 minBS 2 hourBS 4 hourBS 6 hourBS 24 hour1Drug 17:30 AM7:35 AM8:00 AM8:30 AM9:30 AM11:30 AM1:30 PM27:34 AM7:39 AM8:04 AM8:34 AM9:34 AM11:34 AM1:34 PM37:38 AM7:43 AM8:08 AM8:38 AM9:38 AM11:38 AM1:38 PM47:42 AM7:47 AM8:12 AM8:42 AM9:42 AM11:42 AM1:42 PM57:46 AM7:51 AM8:16 AM8:46 AM9:46 AM11:46 AM1:46 PM67:50 AM7:55 AM8:20 AM8:50 AM9:50 AM11:50 AM1:50 PM77:54 AM7:59 AM8:24 AM8:54 AM9:54 AM11:54 AM1:54 PM87:58 AM8:03 AM8:28 AM8:58 AM9:58 AM11:58 AM1:58 PM9Drug 28:02 AM8:07 AM8:32 AM9:02 AM10:02 AM12:02 PM2:02 PM108:06 AM8:11 AM8:36 AM9:06 AM10:06 AM12:06 PM2:06 PM118:10 AM8:15 AM8:40 AM9:10 AM10:10 AM12:10 PM2:10 PM128:14 AM8:19 AM8:44 AM9:14 AM10:14 AM12:14 PM2:14 PM138:18 AM8:23 AM8:48 AM9:18 AM10:18 AM12:18 PM2:18 PM148:22 AM8:27 AM8:52 AM9:22 AM10:22 AM12:22 PM2:22 PM158:26 AM8:31 AM8:56 AM9:26 AM10:26 AM12:26 PM2:26 PM168:35 AM9:00 AM10:30 AM12:30 PM2:30 PM17Drug 38:39 AM9:04 AM10:34 AM12:34 PM2:34 PM188:43 AM9:08 AM10:38 AM12:38 PM2:38 PM198:47 AM9:12 AM10:42 AM12:42 PM2:42 PM208:51 AM9:16 AM10:46 AM12:46 PM2:46 PM218:55 AM9:20 AM10:50 AM12:50 PM2:50 PM228:59 AM9:24 AM10:54 AM12:54 PM2:54 PM239:03 AM9:28 AM10:58 AM12:58 PM2:58 PM249:07 AM9:32 AM11:02 AM1:02 PM3:02 PMRED TIME POINTS are determined as true blood collection times If a smaller amount of blood was necessary, then we could adhere to more time points.BASELINEMice received IP injections of 1 of three “drugs,” (i.e.; saline, or low-dose GOT or high-dose GOT.) Blood samples were taken at various intervals and serum was separated and GOT levels were detected spectrophotometrically. For technical reasons we were only able to successfully measure in the mice receiving the high-dose GOT .Serum levels of GOT remain elevated at 48 hours.
6 Transgenic ALS mice (SOD1-G93A) from The Jackson Laboratory. METHODSTransgenic ALS mice (SOD1-G93A) from The Jackson Laboratory.Weeks of Age Survival EndpointTraining Period(97 – 138 Days of Age)MicereceivedTreatmentbegunTissueCollectionBehavioral TestinggroupedTwenty ALS mice were randomly assigned to treatment groups according to body weight. Mice were singly-housed. The control group received standard chow mash. The DP™ Plan group received The DP™ Plan mixed with standard chow mash. The GOT/OX group received injections of GOT 3X/wk and OX mixed with standard chow mash. The DP + GOT/OX received injections of GOT 3X/wk and OX mixed in a The DP™ Plan-containing chow mash.Treatment Groups1. SD2. SD + DP™ Plan3. SD + GOT/OX4. SD + DP™ Plan + GOT/OX1. SD2. SD + DP3. SD + GOT/OX4. SD + DP + GOT/OX
7 RESULTSAverage daily consumption of energy was unchanged between all groups. However, the SD + The DP™ Plan group showed a decrease in several weight parameters, including total body, liver and lower leg muscle. This suggests a change in the energy utilization of this group. Further investigation into signaling pathway molecules is needed to determine what, if any changes occurred in liver and muscle cells.WeightsSDSD + DPSD + GOT/OXSD + DP + GOT/OXBody (g)23.5 +/- 0.9219.0 +/ **22.2 +/- 0.9720.4 +/- 1.74Liver1084 +/639 +/ *1138 +/1034 +/Muscle111 +/- 8.7463 +/ *90 +/84 +/- 7.05
8 RESULTSThe rotarod is a test used to measure coordination.Mice consuming standard diet experienced more rapid deficits in coordination than mice in treatment groups.
9 RESULTSThe four limb hanging wire test is used to monitor muscle strength and endurance.Mice consuming the The DP™ Plan showed an increase in the preservation of muscle strength relative to all other groups.
10 RESULTS Neurological Score SD+ DP™ Plan , SD+GOT/OX, and SD+GOT/OX+DP attenuate neurological deficit in SOD1-G93A ALS mouse model. Please note that there was no significant difference between treatment groups at baseline measurements (82 days of age) up until 100 days of age. SD+DP™ Plan mice exhibited significantly lower neurological scores (attenuation of hind limb function regression) between days 100 and 115, SD+GOT/OX mice between days 100 and 109, and , and SD+DP™ Plan +GOT/OX mice between days 100 and 111 (p<0.05). SD+DP thus attenuated neurological deficit the longest, followed by SD+GOT/OX, and SD+GOT/OX+DP™ Plan, respectively. All data are represented by means.Note: As the condition of the mouse worsens, its neurological score increases.Criteria of the neurological scoreHinds legs are fully extended during tail elevationHind legs are not fully extended during tail elevation1.5 Abnormal gait2 Toes curl under while walking2.5 Difficulty wqalking using all four legsPulling legs behind their body, but still able to move them3.5 Single hind leg paralysisComplete hind limb paralysis and/or unable to return to upright position within 10 sec.
11 RESULTSMice receiving the SD had shorter survival times than other treatment groups.
12 RESULTS Histology 100 um SD + DP™ Plan SD SD + GOT/OXSD + DP™ PlanSD + DP™ Plan + GOT/OX100 umSoleus muscle cross section with H & E staining. Note “fuller” appearance of the cells in the two frames on the right. These preliminary data suggest that the DP™ Plan may help preserve muscle cell integrity. Further analysis is currently underway in our lab.
13 RESULTS SD SD + DP™ Plan SD + GOT/OX SD + DP™ Plan + GOT/OX Histology Mouse # 571318H&E staining of lumbar sections of mice spinal cords.H&E staining of lumbar sections of mice spinal cords. The anterior horn of the spinal cord contains the descending motor neurons.SD + GOT/OXSD + DP™ Plan + GOT/OX
14 RESULTSHistologyThe Luxol Fast Blue Stain (Myelin Stain) is designed for staining myelin/myelinated axons and Nissil substance on formalin fixed, paraffin-embedded tissue as well as frozen tissue. This product is used for identifying the basic neuronal structure in brain or spinal cord sections.Staining Interpretation:Myelin: BlueNeuropil: PinkNerve Cells: VioletThe Luxol Fast Blue Stain (Myelin Stain) is designed for staining myelin/myelinated axons and Nissil substance on formalin fixed, paraffin-embedded tissue as well as frozen tissue. This product is used for identifying the basic neuronal structure in brain or spinal cord sections. Staining Interpretation: Myelinated Fibers Blue Nissil Substance Violet Nerve Cells VioletSample stained section. We will eventually look at the effects of the different treatments on the cellular make up and or structure of the spinal cord descending motor neurons. myelin fibers appear blue, neuropil appears pink, and nerve cells appear purple.Sample stained section. We will be looking at the effects of the different treatments on the cellular make up and structure of the spinal cord descending motor neurons.
15 CONCLUSIONSCompared to the control group, the three treatment groups each had significantly longer survival times, improved motor performance, and better neurological scores. The treatments were neither additive nor synergistic, suggesting unique therapeutic effects derived from each treatment. suggesting unique therapeutic effects derived from each treatment treatment may have therapeutic application in patients with ALS and perhaps other neurologic disorders.
16 HYPOTHESISOnly about 10% of ALS cases have been shown to have a genetic component. This observation invites speculation as to the etiology of the other 90%. Recent work by Dr. V. Tedone and others suggests a connection with the bacterium genus Borrelia. In this hypothesis, a spirochete of undetermined species is transmitted to the host. A depressed immune system allows the invader access to tissues that are resistant to infiltration by subsequent ordinary dose antibiotics. Flare-ups are explained by bacterial entry into and hijacking of nerve cell metabolism.