A Glutamate Scavenging Protocol Combined with Deanna Protocol in SOD1-G93A Mouse Model of ALS

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive disease of neuronal degeneration in the motor cortex, brainstem, and spinal cord, resulting in impaired motor function and premature demise as a result of insufficient respiratory drive. ALS is associated with dysfunctions in neurons, neuroglia, muscle cells, energy metabolism, and glutamate balance. Currently, there is not a widely accepted, effective treatment for this condition. Prior work from our lab has demonstrated the efficacy of supplemental nutrition with the Deanna Protocol (DP). In the present study, we tested the effects of three different treatments in a mouse model of ALS. These treatments were the DP alone, a glutamate scavenging protocol (GSP) alone, and a combination of the two treatments. Outcome measures included body weight, food intake, behavioral assessments, neurological score, and lifespan. Compared to the control group, DP had a significantly slower decline in neurological score, strength, endurance, and coordination, with a trend toward increased lifespan despite a greater loss of weight. GSP had a significantly slower decline in neurological score, strength, endurance, and coordination, with a trend toward increased lifespan. DP+GSP had a significantly slower decline in neurological score with a trend toward increased lifespan, despite a greater loss of weight. While each of the treatment groups fared better than the control group, the combination of the DP+GSP was not better than either of the individual treatments. We conclude that the beneficial effects of the DP and the GSP in this ALS mouse model are distinct, and appear to offer no additional benefit when combined.

Keywords: CoQ10; Deanna Protocol; amyotrophic lateral sclerosis; blood glutamate scavengers; medium chain triglycerides; motor neuron disease; neurodegeneration; oxaloacetic acid.

Figure 1

Time course of serum GOT levels. Serum was taken at various time intervals following intraperitoneal injections of 5.3 units glutamic oxaloacetate transaminase (GOT) into B6SJLF1/J mice (n = 3 or 4) (Jax Labs stock # 100012). After reaching an apparent maximum at 6 h, elimination occurred with a decay rate of 102.5 h with a T1/2 of 18 h. Data points indicate mean +/− SEM.

Figure 2

Experimental design for survival study. At week 11, behavioral training was initiated. At week 12, treatments were started.

Figure 3

Effect of treatment on body weight. Body weights were measured at weekly intervals—baseline (week 11, when the mice were assigned to treatment groups), Tx (week 12, when treatment began), and every week thereafter until the endpoint. Data points indicate mean +/− SEM. (A) Weight change within groups. The body weight of all groups progressively decreased throughout the course of the experiment. A statistically significant reduction from baseline in the weight of each group (p < 0.05) was noticed by week 15 for group DP+GSP (*), and by week 17 for the three other groups (†). (B) Weight change between groups. The body weights of DP and DP+GSP were significantly less than C at Pre and Endpoint, * p < 0.05. Abbreviations: Tx, Beginning of treatment; Pre, 1 week prior to endpoint.

Figure 4

Average daily food consumption. Food consumption was measured every other day and compared between groups. Data bars with shared alphabetic symbols are not significantly different. Data are presented as mean +/− SEM. (A) There was no difference in the mass of food consumed. (B) There were significant differences between the groups in the energy consumed. Note that the DP+GSP group consumed significantly more daily calories than any of the other groups. Note that the DP group consumed significantly more calories than the C group.

Figure 5

Daily Intake vs. Lifespan. Graphs depicting linear regression of average daily caloric intake versus lifespan. (A) When data from all the groups were plotted, there was a positive correlation between average daily food energy consumed and survival (R² = 0.2565). (B) When data from each group were plotted individually, three of the groups displayed strong positive correlations (R² values for C, DP, and GSP were 0.704, 0.7271, and 0.8898, respectively). However, no such correlation was found for the DP+GSP group (R² = 0.0203).

Figure 6

Effect of treatment on motor function. Graphs depicting average time for each group for each week are shown. Data points indicate mean +/− SEM. After one week of training and familiarization with the accelerating rotarod and the hanging wire test, mice were tested once per week on each apparatus. (A) Rotarod test for endurance and coordination. Compared to baseline, C mice showed a steady decline in performance beginning at week 13, reaching significance at week 17 (p = 0.001). DP mice showed slight statistically insignificant improvement at week 14 before decline began, which reached significance at week 18 (p = 0.004). GSP mice showed a significant improvement at weeks 14 and 15 before decline began, reaching significance at week 18 (p = 0.006). GSP mice performed significantly longer than C at week 14 and 15, indicated by asterisk (p < 0.05). DP+GSP mice remained relatively level up to week 15 before decline began, which reached significance at week 16 (p < 0.05). (B) Hanging wire test for strength. Compared to baseline, C mice showed a decreased performance by week 14. DP mice remained consistent until week 17 and were significantly higher at week 16 than C (p = 0.045). DP mice performed significantly longer than C at week 16, indicated by symbol (p < 0.05). GSP mice remained relatively stable up until week 16. DP+GSP mice remained relatively stable up until week 16.

Figure 7

Effect of treatment on neurological deficit. Graph depicting progression of average neurological score over time. DP, GSP, and DP+GSP attenuate neurological deficit in SOD1-G93A. Note that there was no significant difference between treatment groups at baseline (82 days of age) up until 100 days of age. DP mice exhibited significantly lower neurological scores between days 100 and 115 (indicated by asterisk), SD+GSP mice between days 100–109 and days 113–115 (a total of 13 days), and DP+GSP mice between days 100 and 111 (a total of 12 days) (p < 0.05). DP thus attenuated neurological deficit the longest, followed by GSP and DP+GSP, respectively. All data are represented by means.

Figure 8

Effect of treatment on survival. A Kaplan–Meier survival plot of study groups shows that the DP group shows a trend toward increasing lifespan. No statistically significant differences (p = 0.07). Median survival times (days alive) for each group are also shown.