Identification of deregulated AMPK and mTORC1 signalling in myotonic dystrophy type I and their potential as therapeutic targets

Myotonic Dystrophy type I (DM1) is a disabling multisystemic disease affecting skeletal muscle. The disease is caused by expanded (CTG)n repeats in the 3’UTR of the DMPK gene. (CUG)n-RNA-hairpins formed by the elongated transcripts lead to sequestration of splicing factors, and thereby to mis-splicing of various genes. Although different strategies have been tested to limit splicing defects, no causal treatment is available for this debilitating disease. To better understand the pathophysiology underlying the disease, we analysed whether DM1-associated muscle alterations may be related to a deregulation of central metabolic signalling and/or of the autophagy process in muscle. Although muscle atrophy in DM1 has been previously related to altered signalling and perturbation in catabolic processes, in-depth investigations into these areas are lacking. We showed that muscles from HSALR mice, a well-characterized mouse model for DM1, exhibit a defective response to energy restriction. Mutant muscles reveal blunted AMPK activation under staved conditions, which might be related to splicing-dependent CaMKII deficiency. Additionally, active mTORC1 signalling is maintained in muscle from starved mutant mice, while Akt is efficiently inhibited. We further observed that autophagy flux is impaired in HSALR muscle, which may arise from the deregulation of AMPK-mTORC1 signalling; autophagy is even more severely affected in human DM1 myotubes. Most importantly, normalization of these pathways with pharmacological or dietary approaches improved skeletal muscle strength and significantly reduced myotonia in HSALR mice. In particular, the AMPK agonist, AICAR, but not metformin, another drug known to induce the AMPK pathway, led to a marked amelioration of the relaxation time of mutant muscle, together with partial splicing correction of CLCN1. On the other hand, rapamycin, an mTORC1 inhibitor, and enduring low-protein diet, both reduced myotonia but not DM1-related mis-splicing. Furthermore, mTORC1 inhibition increases muscle force in HSALR mice. Taken together, this suggests that splicing-dependent as well as alternative, splicing-independent mechanisms can improve muscle function in DM1. These findings highlight the involvement of AMPK-mTORC1 imbalance in the disease and illustrate the importance of deregulated cellular processes contributing to DM1 muscle pathophysiology. At the same time this opens new avenues regarding therapeutic options for DM1, by modulating alternative processes aside from RNA toxicity

Targeting deregulated AMPK/mTORC1 pathways improves muscle function in myotonic dystrophy type I

Myotonic dystrophy type I (DM1) is a disabling multisystemic disease that predominantly affects skeletal muscle. It is caused by expanded CTG repeats in the 3'-UTR of the dystrophia myotonica protein kinase (DMPK) gene. RNA hairpins formed by elongated DMPK transcripts sequester RNA-binding proteins, leading to mis-splicing of numerous pre-mRNAs. Here, we have investigated whether DM1-associated muscle pathology is related to deregulation of central metabolic pathways, which may identify potential therapeutic targets for the disease. In a well-characterized mouse model for DM1 (HSALR mice), activation of AMPK signaling in muscle was impaired under starved conditions, while mTORC1 signaling remained active. In parallel, autophagic flux was perturbed in HSALR muscle and in cultured human DM1 myotubes. Pharmacological approaches targeting AMPK/mTORC1 signaling greatly ameliorated muscle function in HSALR mice. AICAR, an AMPK activator, led to a strong reduction of myotonia, which was accompanied by partial correction of misregulated alternative splicing. Rapamycin, an mTORC1 inhibitor, improved muscle relaxation and increased muscle force in HSALR mice without affecting splicing. These findings highlight the involvement of AMPK/mTORC1 deregulation in DM1 muscle pathophysiology and may open potential avenues for the treatment of this disease

mTORC1 and PKB/Akt control the muscle response to denervation by regulating autophagy and HDAC4

Loss of innervation of skeletal muscle is a determinant event in several muscle diseases. Although several effectors have been identified, the pathways controlling the integrated muscle response to denervation remain largely unknown. Here, we demonstrate that PKB/Akt and mTORC1 play important roles in regulating muscle homeostasis and maintaining neuromuscular endplates after nerve injury. To allow dynamic changes in autophagy, mTORC1 activation must be tightly balanced following denervation. Acutely activating or inhibiting mTORC1 impairs autophagy regulation and alters homeostasis in denervated muscle. Importantly, PKB/Akt inhibition, conferred by sustained mTORC1 activation, abrogates denervation-induced synaptic remodeling and causes neuromuscular endplate degeneration. We establish that PKB/Akt activation promotes the nuclear import of HDAC4 and is thereby required for epigenetic changes and synaptic gene up-regulation upon denervation. Hence, our study unveils yet-unknown functions of PKB/Akt-mTORC1 signaling in the muscle response to nerve injury, with important implications for neuromuscular integrity in various pathological conditions

Factors influencing the inhibition of protein kinases

The protein kinase field is a very active research area in the pharmaceutical industry and many activities are ongoing to identify inhibitors of these proteins. The design of new chemical entities with improved pharmacological properties requires a deeper understanding of the factors that modulate inhibitor-kinase interactions. In this report, we study the effect of two of these factors – the magnesium ion cofactor and the protein substrate – on inhibitors of the type I insulin-like growth factor receptor. Our results show that the concentration of magnesium ion influences the potency of ATP competitive inhibitors, suggesting an explanation for the observation that such compounds retain their nanomolar potency in cells despite the presence of millimolar levels of ATP. We also show that the peptidic substrate affects the potency of these IGF1R ATP competitive inhibitors in a different manner, suggesting that the influence of this substrate on compound potency should be taken into consideration during compound optimization