Heterozygous females from a rat model for creatine transporter deficiency reveal altered behavioral response to stressors, normal body weight and slight metabolic changes

Abstract

Creatine (Cr) is an organic acid essential for recycling ATP, important in tissues with high energy demand such as muscle or brain. Cr is synthesized in a 2-step pathway by the enzymes AGAT and GAMT, and transported by SLC6A8 (also called CrT). Cerebral Cr deficiency syndromes (CCDS), due to AGAT, GAMT or CrT deficiencies, are metabolic diseases characterized by brain Cr deficiency, causing a range of clinical features such as severe neurodevelopmental delays and intellectual disability, behavioral disturbances, motor dysfunction and epilepsy. Among CCDS, the X-linked CrT deficiency (CTD) is the most prevalent with no efficient treatment so far. Increasing number of human and animal studies contributes to the understanding of CTD pathology, its diagnosis and treatment, and the roles of Cr and CrT. However, most of them are focused in males and little is known about female carriers and how CrT deficiency affect them. In order to increase knowledge in female sex and roughly explore the relationship with SLC6A8 gene dosage, we present the first characterization of females' Slc6a8 ^Y389C rat model of CTD using both heterozygous and homozygous females. Brain Cr deficiency was found in all homozygous females, while heterozygous ones showed broad variability in brain Cr levels. Elevated and slightly elevated urinary Cr/Crn ratio were present in homozygous and heterozygous females, respectively. Reduced body weight, muscular mass and locomotor activity were hallmarks of homozygous, but not heterozygous, females. However, in contrast to Slc6a8 ^Y389C KI males, spontaneous alternation and grooming behaviors were not affected in any type of Slc6a8 ^Y389C mutant female rats. Interestingly, both Slc6a8 ^Y389C mutant female rats exhibited behavioral abnormalities such as increased prevalence of altered behavioral response to handling, being more frequent in homozygous female rats. Moreover, heterozygous females presented increased anxiety-like behavior to novelty in Open Field Novel Object test and altered behavioral response with increased locomotor activity in response to light as stressor in the Light Dark Box test. These results are coherent with the limited data from CTD human female carriers, validating the Slc6a8 ^Y389C rat females as a promising tool to better understand CTD in female sex. They also provide new insights about CTD pathology, revealing sex and zygotic phenotypic differences, highlighting the importance of including females in the study of CTD.

Keywords: SLC6A8; X-linked disorder; behavior; creatine; creatine transporter deficiency; female; inherited metabolic disease.

Figure 1

Homozygous females show strong CrT deficiency with severe brain creatine deficiency and reduced body weight, while heterozygous females show CrT deficiency with broad effects on brain creatine deficiency and normal body weight. (A) Line-plot showing body weight (in grams) along age (in weeks) per each genotype. Error bars represent standard deviation. No significant differences were found between fWT and fHE rats, but significant differences from 6wo were found between fHO and fWT (as “*”) or between fHO and fHE rats (as ”ª”). 1way-ANOVA and Tukey post hoc; 16 fWT, 21 fHE and 13 fHO. (B) Representative picture of each genotype at 13 weeks-old. Rectangle is 12.5 × 28 cm. (C) Boxplot of urinary Cr/Crn. 15 fWT, 16 fHE and 15 fHO. Pair comparisons were made using Mann-Whitney test with Bonferroni correction. (D) Representative 9.4T ¹H-MRS spectra in the cerebellum of 1 fWT, 2 fHE and 1 fHO. Note the strong decrease of the Cr and PCr peaks in the fHO and the differences in the amplitude of such peaks in fHE. The localization of the measured voxel is presented on top of the panel. (E) Boxplots of ¹H-MRS Cr and/or PCr concentrations in cerebellum (Cb) and hippocampus (Hp) in each genotype. P-values from 1way-ANOVA and pair comparisons using Tukey post hoc test are shown in the table above. 3 fWT, 8 fHE, and 3 fHO. °P-value < 0.1, *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001.

Figure 2

Homozygous, but not heterozygous, female rats show altered muscle phenotype and reduced locomotor activity. (A) Boxplots of muscle Cr and GAA concentrations. Pair comparisons using Mann-Whitney test with Bonferroni correction. 12 fWT, 12 fHE, and 11 fHO. (B) Representative microscopic pictures of hematoxylin/eosin staining in transversal sections of quadriceps muscle. Scale bar = 15 μm. (C) Boxplots of cross-sectional area (CSA) and minimum Feret diameter (Min Feret) from myocytes of quadriceps muscle. Pair comparisons using Mann-Whitney test with Bonferroni correction. 4 fWT, 11 fHE and 4 fHO (128–194 measurements per WT female, 124–281 per fHE, and 156–208 per fHO; 659, 2,000, and 721 total myocytes per genotype, respectively). (D) Barplots of total distance and relative time moving (Mov Duration) in the open field (OF) test. Pair comparisons with Nested ANOVA blocking estrous cycle as a random factor. 11 fWT, 12 fHE, and 11 fHO. (E) Barplots of cumulative duration and frequency of supported and unsupported rearing up (rSupp and rUnsupp, respectively), in the open field (OF) test. Pair comparisons with Nested ANOVA blocking estrous cycle as a random factor. 11 fWT, 12 fHE, and 11 fHO. °P-value < 0.1, *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001.

Figure 3

Mutant females show altered behavioral response to stressors, but similar performance in Y-maze spontaneous alternation test and grooming behavior than WT females. (A) Barplots of cumulative duration (in %), frequency and latency of grooming behavior in an open field. 11 fWT, 12 fHE, and 11 fHO. (B) Y-maze spontaneous alternation test's infographic (left) and barplots (right) showing percentage of alternation, number of entries and distance moved in Y-maze spontaneous alternation test. All genotypes presented significant difference with respect to the value expected by chance in percentage of alternation (100/3, horizontal dot line; p < 0.001, p < 0.001, and p = 0.002 for fWT, fHE, and fHO, respectively, t-test), but no among them. 12 fWT, 12 fHE, and 11 fHO. (C) Barplot of rats' relative distribution presenting (Y) or not (N) behavioral abnormalities in response to handling at the onset of adolescence (7–8 wo). Two-sided Exact binomial test evaluating differences with respect to the fWT group (p < 0.05 and p < 0.001 for fHE and fHO, respectively). 16 fWT, 21 fHE, and 13 fHO. (D) OFNO test's infographic (left) and barplots (right) showing the relative difference in cumulative duration between NO and OF phases spent per each region (C = center, I = intermediate, W = wall; Index Dur) or spent moving (Index Mov), and of the total distance moved in the NO phase (OF phase is shown in Figure 2D). 11 fWT, 10 fHE, and 10 fHO. (E) EPM test's infographic (left) and barplots (right) of cumulative duration spent in each region (CA = closed arm, center, OA = opened arm, HD = head dipping in the OA), and total distance moved. 12 fWT, 12 fHE, and 11 fHO. (F) LDB test's infographic (left) and barplots (right) of cumulative duration spent in the light compartment and average velocity. 12 fWT, 11 fHE, and 8 fHO. Error bars represent standard error of the mean. Pair comparisons among genotypes were performed with Nested ANOVA blocking estrous cycle as a random factor. °P-value < 0.1, *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001.