Medium-Chain Fatty Acids, Beta-Hydroxybutyric Acid and Genetic Modulation of the Carnitine Shuttle Are Protective in a Drosophila Model of ALS Based on TDP-43

Abstract

ALS patients exhibit dyslipidemia, hypermetabolism and weight loss; in addition, cellular energetics deficits have been detected prior to denervation. Although evidence that metabolism is altered in ALS is compelling, the mechanisms underlying metabolic dysregulation and the contribution of altered metabolic pathways to disease remain poorly understood. Here we use a Drosophila model of ALS based on TDP-43 that recapitulates hallmark features of the disease including locomotor dysfunction and reduced lifespan. We performed a global, unbiased metabolomic profiling of larvae expressing TDP-43 (wild-type, TDP^WT or disease-associated mutant, TDP^G298S) and identified several lipid metabolism associated alterations. Among these, we found a significant increase in carnitine conjugated long-chain fatty acids and a significant decrease in carnitine, acetyl-carnitine and beta-hydroxybutyrate, a ketone precursor. Taken together these data suggest a deficit in the function of the carnitine shuttle and reduced lipid beta oxidation. To test this possibility we used a combined genetic and dietary approach in Drosophila. Our findings indicate that components of the carnitine shuttle are misexpressed in the context of TDP-43 proteinopathy and that genetic modulation of CPT1 or CPT2 expression, two core components of the carnitine shuttle, mitigates TDP-43 dependent locomotor dysfunction, in a variant dependent manner. In addition, feeding medium-chain fatty acids or beta-hydroxybutyrate improves locomotor function, consistent with the notion that bypassing the carnitine shuttle deficit is neuroprotective. Taken together, our findings highlight the potential contribution of the carnitine shuttle and lipid beta oxidation in ALS and suggest strategies for therapeutic intervention based on restoring lipid metabolism in motor neurons.

Keywords: TDP-43; amyotrophic lateral sclerosis; beta lipid oxidation; carnitine shuttle; lipid metabolism; metabolomics.

Figure 1

Metabolomic profiling of ALS larvae uncovers deficits in carnitine, carnitine conjugation and lipid beta oxidation. (A) Carnitine shuttle components and fatty acid import into mitochondria. CPT1, 2, carnitine acyltransferases; CACT, carnitine acylcarnitine transferase; IMM, Inner mitochondrial membrane; OMM, Outer mitochondrial membrane. (B) Summary of significantly altered metabolites in TDP^WT and TDP^G298S compared to w¹¹¹⁸ controls. Green indicates significant downregulation and red indicates significant upregulation (P_value < 0.05).

Figure 2

TDP-43 causes alterations in the expression of carnitine shuttle components. (A–C) Transcriptional profiling using q RT PCR of CPT1 mRNA (A), CPT2 mRNA (B) and colt mRNA (C) indicates TDP-43 dependent changes in motor neurons and glia. TDP^WT or TDP^G298S were expressed using D42 GAL4 (motor neurons) or repo GAL4 (glia). Kruskal-Wallis multiple comparisons test was used to calculate significance. n.s., not significant; ^*P_value < 0.05.

Figure 3

Genetic interaction experiments show that CPT1 and CPT2 mitigate locomotor dysfunction caused by TDP^WT and TDP^G298S, in a variant dependent manner. (A) CPT1 knock-down by RNAi (CPT1^RNAi, using y¹v¹; P{y[+t7.7] v[+t1.8] = TRiP.HMS00040}attP2/TM3, Sb¹) mitigates TDP^WT and TDP^G298S larval turning times. (B) CPT2 loss of function (CPT2^f02667, using w¹¹¹⁸; PBac{w[+mC]=WH}CPT2^f02667/TM6B, Tb¹) mitigates TDP^G298S larval turning times. TDP^WT or TDP^G298S were expressed in motor neurons using D42 GAL4. Genotypes as indicated. Kruskal-Wallis multiple comparisons test was used to calculate significance. ^*P_value < 0.05, ^**P_value < 0.01, ^****P_value < 0.0001.

Figure 4

Medium-chain fatty acids and ketones mitigate TDP-43 dependent locomotor dysfunction in a Drosophila model of ALS. (A,B) Larval turning times, used to measure locomotor deficits caused by TDP^WT or TDP^G298S, are rescued by feeding different medium-chain fatty acids [coconut oil (A) or ethyl heptanoate (B)]. (C) Ketone (beta-hydroxybutyrate) feeding reduces larval turning times in ALS larvae. TDP^WT or TDP^G298S were expressed in motor neurons using D42 GAL4. Genotypes and treatments as indicated. Kruskal-Wallis multiple comparisons test was used to calculate significance. ^*P_value < 0.05, ^**P_value < 0.01, ^***P_value < 0.001, ^****P_value < 0.0001.

Figure 5

Model of mitochondrial dysfunction in ALS and proposed interventions. Expression of TDP^WT or TDP^G298S in motor neurons causes alterations in the expression of carnitine shuttle components (ALS mitochondria, B) compared to controls (Healthy mitochondria, A). Blue dashed line for CPT1 indicates an upward trend in transcript levels (P_value = 0.08). These defects can be countered genetically, by reducing CPT1 in the context of TDP^WT (inhibitory blue symbol), or reducing CPT2 in the context of TDP^G298S (inhibitory magenta symbol). Magenta asterisk indicates a potential compensatory mechanism whereby CPT1 RNAi knock-down mitigates TDP^G298S locomotor phenotypes despite CPT1 mRNA being reduced. Alternatively, dietary supplementation with medium-chain fatty acids or beta-hydroxybutyrate bypasses carnitine shuttle dysfunction leading to significant improvement in locomotor function (C, Lipid metabolism interventions in ALS).