Creatine transporter-deficient rat model shows motor dysfunction, cerebellar alterations, and muscle creatine deficiency without muscle atrophy

Abstract

Creatine (Cr) is a nitrogenous organic acid and plays roles such as fast phosphate energy buffer to replenish ATP, osmolyte, antioxidant, neuromodulator, and as a compound with anabolic and ergogenic properties in muscle. Cr is taken from the diet or endogenously synthetized by the enzymes arginine:glycine amidinotransferase and guanidinoacetate methyltransferase, and specifically taken up by the transporter SLC6A8. Loss-of-function mutations in the genes encoding for the enzymes or the transporter cause creatine deficiency syndromes (CDS). CDS are characterized by brain Cr deficiency, intellectual disability with severe speech delay, behavioral troubles, epilepsy, and motor dysfunction. Among CDS, the X-linked Cr transporter deficiency (CTD) is the most prevalent with no efficient treatment so far. Different animal models of CTD show reduced brain Cr levels, cognitive deficiencies, and together they cover other traits similar to those of patients. However, motor function was poorly explored in CTD models, and some controversies in the phenotype exist in comparison with CTD patients. Our recently described Slc6a8^Y389C knock-in rat model of CTD showed mild impaired motor function, morphological alterations in cerebellum, reduced muscular mass, Cr deficiency, and increased guanidinoacetate content in muscle, although no consistent signs of muscle atrophy. Our results indicate that such motor dysfunction co-occurred with both nervous and muscle dysfunctions, suggesting that muscle strength and performance as well as neuronal connectivity might be affected by this Cr deficiency in muscle and brain.

FIGURE 1

Slc6a8 ^xY389C/y KI males present reduced muscular mass and smaller myocytes. A, Urinary Crn (left panel) as well as plasmatic Crn and total CK levels (middle and right panel, respectively) as indicative of the muscular mass. Six WT and six KI: two‐tailed t test. B, Significant decrease of Cr ([μmol/g tissue], two‐tailed t test) and significant increase of GAA levels in muscle ([μmol/g tissue], Mann‐Whitney test) in KI male rats, five WT and five KI. C, Representative macroscopic pictures of WT and KI male hind limbs in frontal/longitudinal (upper panels) and medial/inner lateral views (lower panels) showing reduced volume of quadriceps in KI male rats. D, Representative microscopic pictures of hematoxylin/eosin staining in transversal sections of WT and KI male quadriceps showing significant smaller myocyte diameter in KI males. Scale bar = 15 μm. E, Quantifications of myocyte minimum Feret diameter and cross‐sectional area per each genotype. Mann‐Whitney test, three WT, and three KI (319‐382 measurements per WT and 414‐555 per KI male). Statistical analysis was conducted with R‐3.5.1. Graphs were done using ggplot2 package. Cr, Creatine; Crn, creatinine; GAA, guanidinoacetate; KI, knock‐in; WT, wild type. *P < .05; **P < .01; ***P < 0.001

FIGURE 2

Slc6a8 ^xY389C/y KI males do not show consistent signs of muscle atrophy. A, Muscle expression levels (by quantitative PCR) of Atrogin‐1, Itch, and Gabarapl1 were similar between genotypes and those of Redd1 significantly increased in muscle from KI males in comparison with those of WT males (orange and black boxes, respectively). 18S was used as housekeeping gene for normalization. Four WT and five KI: two‐way ANOVA and Tukey post hoc test. B, Relative levels of phosphorylated S6K1 (pS6K1) and RPS6 (pRPS6) over total S6K1 and RPS6, respectively, were similar between genotypes (left panel). Relative total and phosphorylated protein levels of RPS6 were significantly increased in muscle from KI males while those of S6K1 were similar between genotypes (right panel). Four WT and five KI: two‐way ANOVA and Tukey post hoc test. C, Representative western blot from muscle of WT and KI males. D, Box plots showing no significant differences between genotypes in the area (in μm²), perimeter (in μm), intensity (in % from WT mean value) or the combination of area and intensity (area multiplied by intensity, in % from WT mean value) of individual end plates stained with α‐Bungarotoxin from muscle of WT and KI males. Three WT and three KI using around 35 pictures per animal; Mann‐Whitney test. E, Representative z‐projection confocal images of individual end plates from WT and KI males using α‐Bungarotoxin staining. Scale bar is 5 μm. Statistical analysis was conducted with R‐3.5.1. Graphs were done using ggplot2 package. ANOVA, Analysis of variance; KI, knock‐in; PCR, polymerase chain reaction; WT, wild type. **P < .01; ***P < .001

FIGURE 3

Motor function is affected in Slc6a8 ^xY389C/y KI males. A, In open‐field (OF) test, KI males (orange bars) tended to move less distance with less velocity in comparison with WT males (black bars); in circular corridor (CC) test, KI males moved significantly less distance with less velocity and spent significantly less time moving (Mov) than WT males (gray bars), 10 WT and 10 KI males. B and C, KI males showed significant differences in unsupported (no superior limbs used, B) but not in supported (C) rearing, 9 WT and 10 KI males. Linear mixed models blocking litter as random factor. Statistical analysis was conducted with R‐3.5.1, package lme4. Graphs were done using ggplot2 package. KI, Knock‐in; WT, wild type. *P < .05

FIGURE 4

Cerebellum from Slc6a8 ^xY389C/y KI males is affected. A, Representative confocal z‐projection images of MAP2 (A/B), pNF‐M, NF‐M, and MBP (in red, DAPI in blue) showing decreased immunostaining in KI males. B, Representative confocal z‐projection images of NeuN and Aqp4 immunostainings showing no differences between WT and KI males. C, Representative confocal images of GFAP immunostaining from granular layer (z‐projection, left pictures) and molecular layer (slice of 0.30 μm thickness, right pictures). D, Epifluorescence pictures from cerebellum in sagittal sections showing with white squares the regions in which confocal images were taken. All images correspond to the region labeled in the top picture except the one for MBP immunostaining (in A) which corresponds to the region labeled in the bottom picture. E, Quantification of molecular and granular layers thickness showing a significant reduction in the molecular (m) but not the granular (g) layer of KI males. Mann‐Whitney test, four WT and four KI. Average values from each animal were used for the analysis between genotypes (details in methods), plots showing the variability of all measurements. F, Representative images of Purkinje terminal dendrites from WT and KI males (top and bottom panels, respectively), and quantifications of spines density (left, 101 and 72 measurements from WT and KI, respectively), head diameter (middle, 2024 and 1322 spines from WT and KI, respectively), and spine length (right plot, 2368 and 1491 spines from WT and KI, respectively) showing a significant reduction of the three measurements in KI males. Mann‐Whitney test, four WT and three KI (3‐4 neurons per animal; 20‐30 measurements per rat for spine density, 410‐620 spines per rat for head diameter and spine length). G, Concentration of acetylcholine (Ach), free and total choline (fCho and Cho, respectively) in muscle from WT and KI males, five WT and four KI, t test. Calibration/scale bar in A, B, and C represents 30 μm; 2 μm in F. Statistical analysis was conducted with R‐3.5.1. Graphs were done using ggplot2 package. KI, Knock‐in; MAP2, microtubule‐associated protein 2; MBP, myelin basic protein; NF‐M/pNF‐M, neurofilament mainly present in neuronal soma when dephosphorylated or in axons when phosphorylated; WT, wild type. Mann‐Whitney test, *P < .05; ***P < .001. t test, *P < .05