Emerging evidence has demonstrated that intestinal homeostasis and the microbiome play essential roles in neurological diseases, such as Parkinson's disease. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons and muscle atrophy. Currently, there is no effective treatment. Most patients die within 3–5 years due to respiratory paralysis. Although the death of motor neurons is a hallmark of ALS, other organs may also contribute to the disease progression. We examined the gut of an ALS mouse model, G93A, which expresses mutant superoxide dismutase (SOD1G93A), and discovered a damaged tight junction structure and increased permeability with a significant reduction in the expression levels of tight junction protein ZO-1 and the adherens junction protein E-cadherin. Furthermore, our data demonstrated increased numbers of abnormal Paneth cells in the intestine of G93A mice. Paneth cells are specialized intestinal epithelial cells that can sense microbes and secrete antimicrobial peptides, thus playing key roles in host innate immune responses and shaping the gut microbiome. A decreased level of the antimicrobial peptides defensin 5 alpha was indeed found in the ALS intestine. These changes were associated with a shifted profile of the intestinal microbiome, including reduced levels of Butyrivibrio Fibrisolvens, Escherichia coli, and Fermicus, in G93A mice. The relative abundance of bacteria was shifted in G93A mice compared to wild-type mice. Principal coordinate analysis indicated a difference in fecal microbial communities between ALS and wild-type mice. Taken together, our study suggests a potential novel role of the intestinal epithelium and microbiome in the progression of ALS.

Purpose Emerging evidence has demonstrated that gut microbiome plays essential roles in the pathogenesis of human diseases in distant organs. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons. Treatment with the only FDA approved drug, Riluzole, extends patient life span only for a few months. Thus, there is an urgent need to develop novel interventions for alleviating the disease progression and improving the quality of life for ALS patients. Here we present evidence that intestinal dysfunction and dysbiosis may actively contribute to ALS pathophysiology. Methods We used G93A transgenic mice as a model of human ALS. The G93A mice show abnormal intestinal microbiome and damaged tight junctions, which occur before ALS disease onset. Feeding of the G93A mice with butyrate, a natural bacterial product. Results Feeding of the G93A mice with butyrate, restores the intestinal microbial homeostasis, improves the gut integrity, and prolongs the life span of the ALS mice. Paneth cells are specialized intestinal epithelial cells that regulate the host-bacterial interactions. At the cellular level, we show that abnormal Paneth cells were significantly decreased in the ALS mice treated by butyrate. Butyrate treatment decreases aggregation of the G93A-SOD1 mutated protein in both ALS mice and cultured human intestinal epithelial cells. Implications Our study highlights the complex role of gut microbiome and intestinal epithelium in the progression of ALS and presents butyrate as a potential therapeutic reagent to restore ALS dysbiosis.

SUMMARY Exercise has beneficial effects on metabolism and on tissues. The exercise-induced muscle factor β-aminoisobutyric acid (BAIBA) plays a critical role in the browning of white fat and in insulin resistance. Here we show another function for BAIBA, that of a bone-protective factor that prevents osteocyte cell death induced by reactive oxygen species (ROS). L-BAIBA was as or more protective than estrogen or N-acetyl cysteine, signaling through the Mas-Related G Protein-Coupled Receptor Type D (MRGPRD) to prevent the breakdown of mitochondria due to ROS. BAIBA supplied in drinking water prevented bone loss and loss of muscle function in the murine hindlimb unloading model, a model of osteocyte apoptosis. The protective effect of BAIBA was lost with age, not due to loss of the muscle capacity to produce BAIBA but likely to reduced Mrgprd expression with aging. This has implications for understanding the attenuated effect of exercise on bone with aging.

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We have examined the interaction of transforming growth factor (TGF)␤ receptors with phosphatidylinositol 3-(PI3) kinase in epithelial cells. In COS7 cells, treatment with TGF␤ increased PI3 kinase activity as measured by the ability of p85-associated immune complexes to phosphorylate inositides in vitro. Both type I and type II TGF␤ receptors (T␤R) associated with p85, but the association of T␤RII appeared to be constitutive. The interaction of T␤RI with p85 was induced by treatment with TGF␤. The receptor association with PI3 kinase was not direct as 35 S-labeled rabbit reticulocyte p85 did not couple with fusion proteins containing type I and type II receptors. A kinase-dead, dominant-negative mutant of T␤RII blocked ligand-induced p85-⌻␤RI association and PI3 kinase activity. In T␤RI-null R1B cells, TGF␤ did not stimulate PI3 kinase activity. This stimulation was restored upon reconstitution of T␤RI by transfection. In R1B and NMuMG epithelial cells, overexpression of a dominant active mutant form of T␤RI markedly enhanced ligand-independent PI3 kinase activity, which was blocked by the addition of the T␤RI kinase inhibitor LY580276, suggesting a causal link between T␤RI function and PI3 kinase. Overexpressed Smad7 also prevented ligand-induced PI3 kinase activity. Taken together, these data suggest that 1) TGF␤ receptors can indirectly associate with p85, 2) both receptors are required for ligand-induced PI3 kinase activation, and 3) the activated T␤RI serine-threonine kinase can potently induce PI3 kinase activity.Transforming growth factor ␤ (TGF␤) 1 binds to a heteromeric complex of transmembrane serine-threonine kinases, the type I and II TGF␤ receptors (T␤RI or Alk5 and T␤RII). Following ligand binding to T␤RII, the type I receptor is recruited to the ligand-receptor complex where the constitutively active T␤RII transactivates T␤RI (1). Activated T␤RI phosphorylates the receptor-specific Smad2 and Smad3, which then associate with Smad4 and, as a heteromeric complex, translocate to the nucleus where they regulate the transcription of TGF␤ target genes (1). The Smad signaling pathway mediates the antiproliferative effect of TGF␤ in epithelial cells (2, 3) and is the best characterized. Several non-Smad pathways have also been implicated in mediating the cellular effects of TGF␤. These include the extracellular signal-regulated kinase (Erk), c-Jun N-terminal kinase (Jnk), p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 (PI3) kinase, and the family of Rho GTPases (4, 5).Activated PI3 kinase increases the formation of intracellular 3Ј-phosphorylated inositol lipids, signal transducers that are essential for the regulation of cell cycle progression, glucose metabolism, cell motility, epithelial-to-mesenchymal transition, and apoptosis among others (6, 7). In Swiss 3T3 cells, TGF␤ stimulates PI3 kinase activity, as measured by the ability of immune complexes precipitated with an antibody against p85, the regulatory subunit of PI3 kinase, to induce the formation of phosphatidylinositol-3 mo...

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder characterized by degeneration of motor neurons and atrophy of skeletal muscle. Mutations in the superoxide dismutase (SOD1) gene are linked to 20% cases of inherited ALS. Mitochondrial dysfunction has been implicated in the pathogenic process, but how it contributes to muscle degeneration of ALS is not known. Here we identify a specific deficit in the cellular physiology of skeletal muscle derived from an ALS mouse model (G93A) with transgenic overexpression of the human SOD1 G93A mutant. The G93A skeletal muscle fibers display localized loss of mitochondrial inner membrane potential in fiber segments near the neuromuscular junction. These defects occur in young G93A mice prior to disease onset. Fiber segments with depolarized mitochondria show greater osmotic stress-induced Ca 2؉ release activity, which can include propagating Ca 2؉ waves. These Ca 2؉ waves are confined to regions of depolarized mitochondria and stop propagating shortly upon entering the regions of normal, polarized mitochondria. Uncoupling of mitochondrial membrane potential with FCCP or inhibition of mitochondrial Ca 2؉ uptake by Ru360 lead to cell-wide propagation of such Ca 2؉ release events. Our data reveal that mitochondria regulate Ca 2؉ signaling in skeletal muscle, and loss of this capacity may contribute to the progression of muscle atrophy in ALS.

Limb remote ischemic preconditioning (RIPC) is an effective means of protection against ischemia/reperfusion (IR)–induced injury to multiple organs. Many studies are focused on identifying endocrine mechanisms that underlie the cross-talk between muscle and RIPC-mediated organ protection. We report that RIPC releases irisin, a myokine derived from the extracellular portion of fibronectin domain–containing 5 protein (FNDC5) in skeletal muscle, to protect against injury to the lung. Human patients with neonatal respiratory distress syndrome show reduced concentrations of irisin in the serum and increased irisin concentrations in the bronchoalveolar lavage fluid, suggesting transfer of irisin from circulation to the lung under physiologic stress. In mice, application of brief periods of ischemia preconditioning stimulates release of irisin into circulation and transfer of irisin to the lung subjected to IR injury. Irisin, via lipid raft–mediated endocytosis, enters alveolar cells and targets mitochondria. Interaction between irisin and mitochondrial uncoupling protein 2 (UCP2) allows for prevention of IR-induced oxidative stress and preservation of mitochondrial function. Animal model studies show that intravenous administration of exogenous irisin protects against IR-induced injury to the lung via improvement of mitochondrial function, whereas in UCP2-deficient mice or in the presence of a UCP2 inhibitor, the protective effect of irisin is compromised. These results demonstrate that irisin is a myokine that facilitates RIPC-mediated lung protection. Targeting the action of irisin in mitochondria presents a potential therapeutic intervention for pulmonary IR injury.

Defective coupling between sarcoplasmic reticulum and mitochondria during control of intracellular Ca 2؉ signaling has been implicated in the progression of neuromuscular diseases. Our previous study showed that skeletal muscles derived from an amyotrophic lateral sclerosis (ALS) mouse model displayed segmental loss of mitochondrial function that was coupled with elevated and uncontrolled sarcoplasmic reticulum Ca 2؉ release activity. The localized mitochondrial defect in the ALS muscle allows for examination of the mitochondrial contribution to Ca 2؉ removal during excitation-contraction coupling by comparing Ca 2؉ transients in regions with normal and defective mitochondria in the same muscle fiber. Here we show that Ca 2؉ transients elicited by membrane depolarization in fiber segments with defective mitochondria display an ϳ10% increased amplitude. These regional differences in Ca 2؉ transients were abolished by the application of 1,2-bis(O-aminophenoxy)ethane-N,N,N,N-tetraacetic acid, a fast Ca 2؉ chelator that reduces mitochondrial Ca 2؉ uptake. Using a mitochondria-targeted Ca 2؉ biosensor (mt11-YC3.6) expressed in ALS muscle fibers, we monitored the dynamic change of mitochondrial Ca 2؉ levels during voltage-induced Ca 2؉ release and detected a reduced Ca 2؉ uptake by mitochondria in the fiber segment with defective mitochondria, which mirrored the elevated Ca 2؉ transients in the cytosol. Our study constitutes a direct demonstration of the importance of mitochondria in shaping the cytosolic Ca 2؉ signaling in skeletal muscle during excitation-contraction coupling and establishes that malfunction of this mechanism may contribute to neuromuscular degeneration in ALS.