Senataxin mutations elicit motor neuron degeneration phenotypes and yield TDP-43 mislocalization in ALS4 mice and human patients.

Amyotrophic lateral sclerosis type 4 (ALS4) is a rare, early-onset, autosomal dominant form of ALS, characterized by slow disease progression and sparing of respiratory musculature. Dominant, gain-of-function mutations in the senataxin gene (SETX) cause ALS4, but the mechanistic basis for motor neuron toxicity is unknown. SETX is a RNA-binding protein with a highly conserved helicase domain, but does not possess a low-complexity domain, making it unique among ALS-linked disease proteins. We derived ALS4 mouse models by expressing two different senataxin gene mutations (R2136H and L389S) via transgenesis and knock-in gene targeting. Both approaches yielded SETX mutant mice that develop neuromuscular phenotypes and motor neuron degeneration. Neuropathological characterization of SETX mice revealed nuclear clearing of TDP-43, accompanied by TDP-43 cytosolic mislocalization, consistent with the hallmark pathology observed in human ALS patients. Postmortem material from ALS4 patients exhibited TDP-43 mislocalization in spinal cord motor neurons, and motor neurons from SETX ALS4 mice displayed enhanced stress granule formation. Immunostaining analysis for nucleocytoplasmic transport proteins Ran and RanGAP1 uncovered nuclear membrane abnormalities in the motor neurons of SETX ALS4 mice, and nuclear import was delayed in SETX ALS4 cortical neurons, indicative of impaired nucleocytoplasmic trafficking. SETX ALS4 mice thus recapitulated ALS disease phenotypes in association with TDP-43 mislocalization and provided insight into the basis for TDP-43 histopathology, linking SETX dysfunction to common pathways of ALS motor neuron degeneration.

Differential effects of various genetic mouse models of the mechanistic target of rapamycin complex I inhibition on heart failure.

Inhibition of mammalian target of rapamycin complex I (mTORC1) by rapamycin improves cardiac function in both aging and heart failure. While the protective mechanisms are not fully understood in mammals, they are presumably mediated through metabolic regulation and suppression of protein translation by reduced phosphorylation of 4EBP1, a target of mTORC1. Using transverse aortic constriction (TAC) and Gαq overexpression-induced heart failure models, we examined the effect of cardiac-specific heterozygous deletion (het) of Raptor, a component of mTORC1, and cardiac-specific transgenic overexpression of wild type or phosphorylation site mutant 4EBP1. In wild-type mice with TAC-induced heart failure, quantitative shotgun proteomics revealed decreased abundance of proteins of mitochondrial metabolism and increased abundance of proteins in oxidative stress response, ubiquitin, and other pathways. The Raptor het ameliorated both TAC- and Gαq overexpression-induced heart failure and the associated proteomic remodeling, especially those pathways involved in mitochondrial function, citric acid cycle, and ubiquitination. In contrast, transgenic overexpression of either wild type or mutant 4EBP1 aggravated TAC and Gαq, consistent with reduced adaptive hypertrophy by suppression of protein translation, in parallel with adverse remodeling of left ventricular proteomes. Partial mTORC1 inhibition by Raptor heterozygous deletion ameliorates heart failure and is associated with better preservation of the mitochondrial proteome; however, this effect does not appear to be mediated through suppression of protein translation by increased 4EBP1. Increased activity of 4EBP1 reduced adaptive hypertrophy and aggravated heart failure, suggesting that protein translation is essential for adaptive hypertrophy in pressure overload.

Increased 4E-BP1 Expression Protects against Diet-Induced Obesity and Insulin Resistance in Male Mice.

Obesity is a major risk factor driving the global type II diabetes pandemic. However, the molecular factors linking obesity to disease remain to be elucidated. Gender differences are apparent in humans and are also observed in murine models. Here, we link these differences to expression of eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), which, upon HFD feeding, becomes significantly reduced in the skeletal muscle and adipose tissue of male but not female mice. Strikingly, restoring 4E-BP1 expression in male mice protects them against HFD-induced obesity and insulin resistance. Male 4E-BP1 transgenic mice also exhibit reduced white adipose tissue accumulation accompanied by decreased circulating levels of leptin and triglycerides. Importantly, transgenic 4E-BP1 male mice are also protected from aging-induced obesity and metabolic decline on a normal diet. These results demonstrate that 4E-BP1 is a gender-specific suppressor of obesity that regulates insulin sensitivity and energy metabolism.

Muscle-specific 4E-BP1 signaling activation improves metabolic parameters during aging and obesity.

Eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) is a key downstream effector of mTOR complex 1 (mTORC1) that represses cap-dependent mRNA translation initiation by sequestering the translation initiation factor eIF4E. Reduced mTORC1 signaling is associated with life span extension and improved metabolic homeostasis, yet the downstream targets that mediate these benefits are unclear. Here, we demonstrated that enhanced 4E-BP1 activity in mouse skeletal muscle protects against age- and diet-induced insulin resistance and metabolic rate decline. Transgenic animals displayed increased energy expenditure; altered adipose tissue distribution, including reduced white adipose accumulation and preserved brown adipose mass; and were protected from hepatic steatosis. Skeletal muscle-specific 4E-BP1 mediated metabolic protection directly through increased translation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and enhanced respiratory function. Non-cell autonomous protection was through preservation of brown adipose tissue metabolism, which was increased in 4E-BP1 transgenic animals during normal aging and in a response to diet-induced type 2 diabetes. Adipose phenotypes may derive from enhanced skeletal muscle expression and secretion of the known myokine FGF21. Unlike skeletal muscle, enhanced adipose-specific 4E-BP1 activity was not protective but instead was deleterious in response to the same challenges. These findings indicate that regulation of 4E-BP1 in skeletal muscle may serve as an important conduit through which mTORC1 controls metabolism.