TML, GBB, and carnitine levels were substantially changed in both the plasma and brain tissues of Tmlhe-KO compared to those in their WT counterparts (Fig. 1), which is in line with the predicted functionality of the TMLD enzyme. We found up to 3.5-fold higher TML concentrations in plasma (1.51 ± 0.02 KO vs. 0.62 ± 0.10 WT nmol/ml) and brain tissue (37.50 ± 0.65 KO vs. 9.86 ± 0.26 WT nmol/g) of Tmlhe-KO mice than those of WT mice. GBB is an important intermediate metabolite in the carnitine synthesis pathway [16]. Since GBB was not detectable in Tmlhe-KO mouse plasma and brain tissues, we concluded that the carnitine synthesis pathway was severely interrupted in these animals. As a result, carnitine concentrations in Tmlhe-KO mouse plasma (0.92 ± 0.07 KO vs. 26.50 ± 1.93 WT nmol/l) and brain tissue (10.91 ± 0.39 KO vs. 121.1 ± 4.44 WT nmol/g) were significantly lowered to 90% of those in WT mice. Alterations in TML, GBB, and carnitine concentrations were observed in both male and female Tmlhe-KO mice. The Tmlhe-KO mice fully mimicked the most severe manifestations of inherited TMLHE gene deficiency in humans according to the carnitine, GBB, and TML levels.

TML, GBB, and carnitine concentrations in WT and Tmlhe-KO mouse brains (A) and plasma (B). Concentrations of GBB and carnitine were significantly lower in KO mice than in WT mice. Each value is the mean (± SEM) of 5–8 mice. **p < 0.0001 compared to the WT group (two-way ANOVA followed by unpaired t-test). Coresponding F and p values are: plasma TML F3, 16 = 28.48, p < 0.0001; plasma GBB F3, 16 = 22.62, p < 0.0001; plasma carnitine F3, 16 = 168.6, p < 0.0001; brain TML F3, 25 = 741.5, p < 0.0001; brain GBB F3, 24 = 457.6; p < 0.0001, brain carnitine F3, 25 = 479.6; p < 0.0001

We also compared the performance of the WT and Tmlhe-KO mice in a range of social behavior and motor function tests. In the three-chamber social test, Tmlhe-KO female and male mice displayed robust sociability by spending more time sniffing the novel mouse than the novel object (Table 1). In the marble-burying test, we observed that all mice were interested in marbles; there was no difference in marble burying between WT and Tmlhe-KO mice (Table 1). Nest-building behavior in the nest-building test also was unchanged in Tmlhe-KO mice (Table 1). Furthermore, Tmlhe-KO female mice demonstrated normal maternal behavior, including licking of pups, nest building, and crouching over grouped pups. We also tested mouse grip strength and performance on the accelerating rotarod test because both carnitine deficiency and ASD are associated with impairments in muscle strength and motor coordination [3]. The forelimb grip strength and rotarod performance of both male and female Tmlhe-KO mice was similar to WT mice, indicating that muscle strength and coordination in Tmlhe-KO mice were fully preserved despite very low carnitine concentrations (Table 1, Fig. 1). Patients with ASD might have variable degrees of intellectual disability [13]; therefore, we examined both working and contextual memory in mice using the Y-maze and passive-avoidance tests, respectively. We did not observe any differences in cognitive function between Tmlhe-KO mice and WT mice (Table 1). These results also confirmed that low levels of carnitine did not induce behavioral changes characteristic of ASD.

ASD has been associated with mitochondrial energy failure [3]. A retrospective study reported that 5% (vs. 0.01% in control) of children with ASD had a mitochondrial disease that was determined by abnormal concentrations of biomarkers such as increased plasma levels of lactate, alanine and acylcarnitines, which were suggested as markers of mitochondrial dysfunction [3]. The alanine plasma concentration was not increased in the Tmlhe-KO mice, but concentrations of lactate and acylcarnitines were significantly decreased, suggesting more efficient utilization of energy metabolism substrates in these mice [11]. Compared to WT mice, Tmlhe-KO mice exhibited improved mitochondrial functionality, particularly the OXPHOS-dependent respiration rate and OXPHOS coupling efficiency, in several tissues [11]. In a previous study, we reported that mitochondria in Tmlhe-KO mice are better protected against ischemia‒reperfusion-induced damage [11]. In contrast to OCTN2-deficient JVS mice [7, 8], signs of cardiac, muscle, and liver dysfunction were not observed over the 2-year lifespan of Tmlhe-KO mice, similar to WT mice (Table 1) [11]. This suggests that the transporter functions of carnitine or other organic cations might be more important for mitochondrial functionality than low levels of carnitine alone.

Despite the robust findings in the Tmlhe-KO mouse model, patients can be more sensitive to very low carnitine levels.