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{"project":"2_test","denotations":[{"id":"23785402-18000159-91676670","span":{"begin":171,"end":173},"obj":"18000159"},{"id":"23785402-17522299-91676671","span":{"begin":443,"end":445},"obj":"17522299"},{"id":"23785402-20079427-91676672","span":{"begin":968,"end":970},"obj":"20079427"},{"id":"23785402-12401889-91676673","span":{"begin":1746,"end":1748},"obj":"12401889"},{"id":"23785402-17438338-91676674","span":{"begin":2148,"end":2150},"obj":"17438338"},{"id":"23785402-21934272-91676675","span":{"begin":3183,"end":3185},"obj":"21934272"},{"id":"23785402-15907797-91676676","span":{"begin":3294,"end":3296},"obj":"15907797"},{"id":"23785402-21934272-91676677","span":{"begin":4951,"end":4953},"obj":"21934272"},{"id":"23785402-21841792-91676678","span":{"begin":6092,"end":6094},"obj":"21841792"},{"id":"23785402-2027048-91676679","span":{"begin":9345,"end":9347},"obj":"2027048"},{"id":"23785402-19404401-91676680","span":{"begin":10873,"end":10874},"obj":"19404401"},{"id":"23785402-20004097-91676681","span":{"begin":11233,"end":11235},"obj":"20004097"}],"text":"Results\n\nSCD1 expression is altered in ALS muscle\nOn the basis of our previous microarray data, obtained from a transgenic mouse model of mutant SOD1-linked familial ALS [11], in this study we investigated the significance of the down-regulation of SCD1 for the metabolic capacity of muscles and their response to injury. The expression of SCD1 in the gastrocnemius of SOD1(G86R) mice, which are affected by a progressive denervation atrophy [19], was already diminished at 60 days of age. In this respect, it is noteworthy to mention that our previous electromyography studies on this mouse line revealed that the amplitudes of the compound muscle action potentials, a reduction of which typically reflects a decrease in the number of functional motor units, were normal at the age of 75 days. In addition, mice did not present at this age any abnormal spontaneous electrical activity, which would have reflected the common response of muscle to loss of innervation [16]. According to these findings, we can conclude that SCD1 down-regulation occurred precociously in our SOD1(G86R) mouse model. We then showed here that the decrease in SCD1 expression also persisted during the course of the disease, at 90 days of age, when muscle denervation becomes detectable and motor deficits usually arise, and at about 105 days of age, when hind legs start to be paralysed. At that moment, the decrease in SCD1 expression was also noticeable in the tibialis anterior, which is another muscle in the mouse hind leg displaying less oxidative metabolism than the gastrocnemius (Figure 1A). As a consequence of the repression of muscle SCD1 expression, we observed that the C18:1/C18:0 fatty acid ratio, an index of the desaturation activity of the enzyme [20], was slightly reduced in presymptomatic muscle extracts but significantly diminished at the end stage in both gastrocnemius and tibialis anterior (Figure 1B). It is noteworthy to mention that our previous studies had shown that SOD1(G86R) mice typically exhibit decreased postprandial lipidemia and increased peripheral clearance of lipids, both of which can be ascribed to muscle hypermetabolism [10]. Therefore, an excess of uptake of exogenous lipids in this tissue could mask otherwise earlier and more robust differences in the index of SCD activity.\n10.1371/journal.pone.0064525.g001 Figure 1 SCD1 expression and activity in ALS mouse muscle.\n(A) Time course of SCD1 expression in gastrocnemius (GT, brown columns) and tibialis anterior (TA, orange columns) from SOD1(G86R) mice at indicated ages. Wild-type expression is represented by 100% baseline. ***P\u003c0.001 (One sample t-test, n = 5–11). (B) C18:1/C18:0 fatty acid ratio in gastrocnemius and tibialis anterior from SOD1(G86R) mice (brown columns) and wild-type littermates (white columns) at indicated ages. *P\u003c0.05 (1-way ANOVA followed by Bonferroni's multiple comparison test for gastrocnemius, and unpaired t-test for tibialis anterior, n = 3–10). To obtain independent evidence that SCD1 down-regulation is a typical feature of ALS, we took advantage of our transcriptome database composed of deltoid biopsies from patients with the sporadic form of the disease [12]. The expression of not only SCD1 but also SCD5, a primate-specific enzyme variant with identical function [21], was lower in ALS patients, as compared to normal control subjects. Furthermore, the repression of SCD1 expression was much more remarkable in a muscle not clinically or electromyography affected than in a muscle at an advanced stage of pathology, characterized at the clinical level by reduced strength and neurogenic electromyography pattern (Figure 2A). That SCD1 down-regulation could be observed both in presymptomatic SOD1(G86R) mouse muscle and in relatively healthy human ALS muscle prompted us to speculate that such a pattern of expression might not be solely related to the loss of muscle innervation characteristic of the disease. To address this question, we compared SCD1 expression in gastrocnemius submitted to acute denervation, as obtained by cutting and removing several millimeters of the sciatic nerve, or transient denervation followed by re-innervation, as obtained by crushing the sciatic nerve for several seconds. Under these conditions, the expression of SCD1 was increased after axotomy but significantly reduced after crush (Figure 2B). Overall, these findings provide evidence for the implication of SCD1 in the pathological process triggering ALS, and suggest that SCD1 down-regulation could be involved in the restoration of muscle function in response to injury.\n10.1371/journal.pone.0064525.g002 Figure 2 SCD1 expression in ALS patient muscle and after nerve injury.\n(A) Expression of SCD1 and SCD5 in deltoid muscle biopsies from ALS patients and healthy subjects (CT, white columns), as identified by microarray analysis of the database deposited at http://www.ebi.ac.uk/arrayexpress/(accession number E-MEXP-3260) [12]. ALS samples were obtained from muscle not clinically or electromyography affected (Unaff, orange columns) and from muscle with advanced pathology, characterized by reduced strength and neurogenic electromyography pattern (Aff, brown columns). *P\u003c0.05 (1-way ANOVA followed by Tukey's multiple comparison test, n = 4–10). (B) Expression of SCD1 in gastrocnemius following sciatic nerve axotomy (Axo) or crush at indicated post-operation days. Contralateral muscle expression is represented by 100% baseline. **P\u003c0.01, ***P\u003c0.001 (One sample t-test, n = 4–10).\n\nSCD1 knockout mice do not manifest motor impairment but display exacerbated muscle metabolic oxidative capacity\nTo gain insight into the way in which the lack of SCD1 expression impacts on muscle function, we investigated several characteristics of muscles in these SCD1 knockout mice reflecting their metabolic status, and also evaluated their motor behavior. At the molecular level, we measured the expression of PGC1-α, PPARα and PDK4, of which an increase is involved in stimulating mitochondrial biogenesis and in switching the energy source from glucose to fatty acids [22]. The expression of these genes was significantly higher in the gastrocnemius of SCD1 knockout mice, as compared to wild-type littermates; in the tibialis anterior, there was also a trend toward an increased expression (Figure 3A). Despite this latter attenuated response, the tibialis anterior represents, better than the gastrocnemius, a typical example of glycolytic muscle in which to evaluate changes in the relative density of the various fiber types. We therefore used this muscle to determine potential morphological and biochemical changes triggered by the absence of SCD1. The number of fibers per muscle section was higher in SCD1 knockout mice than in wild-type mice (Figure 3B). Accordingly, the distribution of fiber calibers showed an increase in the amount of fibers of small caliber in SCD1 knockout mice (Figure 3C). We extended these findings by performing SDH histochemistry, and found that the average cross-sectional area of both SDH-positive and SDH-negative fibers was smaller in SCD1 knockout mice than in wild-type mice (Figure 3D). These differences were associated in SCD1 knockout mice with a significant predominance of SDH-positive fibers, which are characterized by a higher metabolic oxidative capacity (Figure 3E).\n10.1371/journal.pone.0064525.g003 Figure 3 Metabolic phenotype of muscle from SCD1 knockout mice.\n(A) Expression of PGC1-α, PPARα and PDK4 in gastrocnemius and tibialis anterior from SCD1 knockout mice (brown columns) and wild-type littermates (white columns). *P\u003c0.05, **P\u003c0.01 (Unpaired t-test, n = 3–11). (B) Number of muscle fibers in tibialis anterior from SCD1 knockout mice (KO, brown column) and wild-type littermates (WT, white column). *P\u003c0.05 (Unpaired t-test, n = 7–10). (C) Distribution of the calibers of muscle fibers in tibialis anterior from SCD1 knockout mice (327 fibers, black circles) and wild-type littermates (283 fibers, white circles). Representative microphotographs of wild-type and knockout tibialis anterior are shown. (D) Averaged cross-sectional area of SDH-positive and SDH-negative fibers in tibialis anterior from SCD1 knockout mice (brown columns) and wild-type littermates (white columns). *P\u003c0.05, **P\u003c0.01 (Unpaired t-test, n = 7–10). (E) Number of SDH-positive (orange bars) and SDH-negative fibers (white bars) in tibialis anterior from SCD1 knockout mice (KO) and wild-type littermates (WT). ***P\u003c0.001 (Chi-square test, n = 283–327). Evaluation of muscle function using the grip strength test revealed no changes in the force developed by hind limbs between SCD1 knockout mice and their wild-type littermates (0.37±0.024 N in SCD1 knockout mice versus 0.37±0.021 N in wild-type mice, n = 7). Along with this, no abnormal spontaneous electrical activity, which would have reflected the typical response of muscle to loss of innervation, was found in the gastrocnemius of SCD1 knockout mice (data not shown). In contrast, we also measured the expression of a series of genes specific to the motor end plate, including the acetylcholine receptor subunits α, γ and ε (AChR-α, AChR-γ and AChR-ε, respectively), and muscle-specific receptor tyrosine kinase (MuSK). Except for AChR-γ, of which an increase would have been considered a sign of muscle denervation [23], the expression of these genes was significantly increased in the gastrocnemius of SCD1 knockout mice as compared to their wild-type littermates, although the changes were less pronounced in the tibialis anterior (Figure 4). In all, these results indicate that the genetic ablation of SCD1 is not detrimental per se to muscle function but promotes a metabolic shift toward a more oxidative capacity, and stimulates the neuromuscular junction gene expression program.\n10.1371/journal.pone.0064525.g004 Figure 4 Gene expression specific to the motor end plate in SCD1 knockout mice.\nExpression of AChR-α, AChR-γ, AChR-ε and MuSK in gastrocnemius and tibialis anterior from SCD1 knockout mice (brown columns) and wild-type littermates (white columns). *P\u003c0.05, ***P\u003c0.001 (Unpaired t-test, n = 4–11).\n\nSCD deficiency accelerates muscle function recovery after nerve injury\nAs shown above, the lack of SCD1 expression does not represent a handicap for muscle function. Therefore, its down-regulation, as observed in ALS or after nerve crush, prompts us to hypothesize that the enzyme may participate in the restorative efforts that muscles experience at the early stages of disease, when neuromuscular deterioration is not generalized yet, or during the process of recovery following a brief disruption of the neuromuscular communication. To address this question, we took advantage of such a model of transient denervation and re-innervation as a means to evaluate, by performing relatively manageable short-term experiments [3], the importance of SCD1 for the restoration of muscle function in response to nerve damage. We performed these experiments using not only SCD1 knockout mice but also mice deficient in SCD enzymatic activity, as obtained by feeding them with MF-438, which is an orally bioavailable pharmacological agent inhibiting SCD-dependent desaturation of fatty acids [17]. To verify if our treatment was biologically active in vivo, we measured several parameters that should reflect the deficiency in SCD enzymatic activity. First, MF-438 significantly reduced both C16:1/C16:0 and C18:1/C18:0 fatty acid ratios in circulating lipids (Figure 5A). Second, MF-438 also induced a decrease in the respiratory quotient as determined by indirect calorimetry, which indicated a switch of the energy source from glucose to fatty acids (Figure 5B). Finally, the drug triggered concomitantly a small but significant decrease in body mass of mice treated for two weeks (Figure 5C). SCD deficiency did not alter hind limb grip strength during the 2-week treatment, as determined either by a percentage of peak force at day 0 (Figure 5D) or by normalizing peak force to body mass (Figure 5E). Also, there were no detectable electromyography abnormalities suggestive of denervation (data not shown). Furthermore, MF-438 stimulated the expression of several genes specific to the motor end plate (Figure 5F). Overall, these findings strongly support that the effects of the SCD inhibitor are very similar to those observed in SCD1 knockout mice.\n10.1371/journal.pone.0064525.g005 Figure 5 Effects of MF-438 on metabolism and muscle function.\n(A) C16:1/C16:0 and C18:1/C18:0 fatty acid ratio in plasma from MF-438 treated mice (brown columns) and control littermates (CT, white columns). ***P\u003c0.001 (Unpaired t-test, n = 4–6). (B) Time course of respiratory quotient (RQ) before and after treatment with MF-438 at a dose of 10 mg/kg body mass/day (indicated by the black bar) (n = 4). Time course of body mass (C), muscle grip strength expressed as a percentage of day 0 (D), and specific grip strength, as determined by normalizing peak force to body mass (E), in mice fed regular chow (white circles) and mice fed regular chow supplemented with MF-438 (black circles). **P\u003c0.01 (2-way ANOVA followed by Bonferroni test, n = 5–6). (F) Expression of PGC1-α, AChR-α, and MuSK in gastrocnemius from MF-438 treated mice (brown columns) and control littermates (white columns). *P\u003c0.05 (Unpaired t-test, n = 3–9). To monitor the recovery of muscle function after crushing the sciatic nerve, we measured hind limb grip strength during a post-lesion period of two weeks, and found that the force in SCD1 knockout mice was restored to its initial level more rapidly than in their wild-type littermates. Accordingly, the proportion of abnormal electromyography episodes reflecting neurogenic muscle denervation was lower in SCD1 knockout mice at 14 days post-lesion (Figure 6A). We also measured at that time the relative density of muscle fiber types as a witness to the restorative process. In the denervated tibialis anterior of wild-type mice, the proportion of fibers intensely stained by SDH histochemistry (presumably, slow-twitch type I fibers) was very low, and there was a significant predominance of medium-stained fibers (presumed fast-twitch fatigue-resistant type IIA fibers). In contrast, the distribution of fiber types in the denervated tibialis anterior of SCD1 knockout mice was identical to that observed in the muscle contralateral to the lesion (Figure 6B), suggesting the establishment of a normal non-stressed situation. To corroborate these findings, we followed muscle function recovery after crush in MF-438 treated mice, and also found accelerated, though not complete, restoration of grip strength, as well as reduced extent of electromyography abnormalities (Figure 6C). Furthermore, quantification of the percentage of mice that, after initial total paralysis, started to exhibit a grip strength distinct from zero showed that on average the recovery in treated mice took place three days sooner than in untreated mice (Figure 6D). In all, these results strongly suggest that reducing SCD enzymatic activity stimulates the restorative potential of skeletal muscles.\n10.1371/journal.pone.0064525.g006 Figure 6 Muscle function recovery in SCD-deficient mice submitted to nerve crush.\nRestoration of muscle grip strength in SCD1 knockout mice (A) or MF-438 treated mice (C) (black circles) and corresponding control littermates (white circles) at the indicated post-operation times. ***P\u003c0.001 (2-way ANOVA, n = 4–12). Percentage of electromyography episodes of spontaneous activity in SCD1 knockout mice (Inset A) or MF-438 treated mice (Inset C) (KO or MF, brown columns) and corresponding control littermates (WT or CT, white columns) two weeks after sciatic nerve crush. *P\u003c0.05 (Unpaired t-test, n = 4–7). (B) Relative density of muscle fiber types in ipsilateral and contralateral tibialis anterior from SCD1 knockout mice and wild-type littermates two weeks after sciatic nerve crush. According to SDH histochemistry, fibers were classified as dark brown colored fibers with high metabolic oxidative capacity (brown columns), pale brown colored fibers with medium oxidative capacity (orange columns) and non-satined fibers (white columns). *P\u003c0.05 and ***P\u003c0.001 (1-way ANOVA followed by Tukey's multiple comparison test, (n = 4–6). (D) Kaplan-Meier curves showing the percentage of MF-438 treated mice (black circles) and control littermates (white circles) that started to exhibit a grip strength distinct from zero after initial total paralysis. Logrank test (n = 10–11). Inset D, averaged time at start of recovery in MF-438 treated mice (MF, brown column) and control littermates (CT, white column). *P\u003c0.05 (Unpaired t-test, n = 10–11).\n\nDi"}

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