Distinct transcriptomic profile of satellite cells contributes to preservation of neuromuscular junctions in extraocular muscles of ALS mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder characterized by progressive weakness of almost all skeletal muscles, whereas extraocular muscles (EOMs) are comparatively spared. While hindlimb and diaphragm muscles of end-stage SOD1G93A (G93A) mice (a familial ALS mouse model) exhibit severe denervation and depletion of Pax7+satellite cells (SCs), we found that the pool of SCs and the integrity of neuromuscular junctions (NMJs) are maintained in EOMs. In cell sorting profiles, SCs derived from hindlimb and diaphragm muscles of G93A mice exhibit denervation-related activation, whereas SCs from EOMs of G93A mice display spontaneous (non-denervation-related) activation, similar to SCs from wild-type mice. Specifically, cultured EOM SCs contain more abundant transcripts of axon guidance molecules, including Cxcl12, along with more sustainable renewability than the diaphragm and hindlimb counterparts under differentiation pressure. In neuromuscular co-culture assays, AAV-delivery of Cxcl12 to G93A-hindlimb SC-derived myotubes enhances motor neuron axon extension and innervation, recapitulating the innervation capacity of EOM SC-derived myotubes. G93A mice fed with sodium butyrate (NaBu) supplementation exhibited less NMJ loss in hindlimb and diaphragm muscles. Additionally, SCs derived from G93A hindlimb and diaphragm muscles displayed elevated expression of Cxcl12 and improved renewability following NaBu treatment in vitro. Thus, the NaBu-induced transcriptomic changes resembling the patterns of EOM SCs may contribute to the beneficial effects observed in G93A mice. More broadly, the distinct transcriptomic profile of EOM SCs may offer novel therapeutic targets to slow progressive neuromuscular functional decay in ALS and provide possible 'response biomarkers' in pre-clinical and clinical studies.

Distinct transcriptomic profile of satellite cells contributes to preservation of neuromuscular junctions in extraocular muscles of ALS mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder characterized by progressive weakness of almost all skeletal muscles, whereas extraocular muscles (EOMs) are comparatively spared. While hindlimb and diaphragm muscles of end-stage SOD1G93A (G93A) mice (a familial ALS mouse model) exhibit severe denervation and depletion of Pax7 + satellite cells (SCs), we found that the pool of SCs and the integrity of neuromuscular junctions (NMJs) are maintained in EOMs. In cell sorting profiles, SCs derived from hindlimb and diaphragm muscles of G93A mice exhibit denervation-related activation, whereas SCs from EOMs of G93A mice display spontaneous (non-denervation-related) activation, similar to SCs from wild-type mice. Specifically, cultured EOM SCs contain more abundant transcripts of axon guidance molecules, including Cxcl12 , along with more sustainable renewability than the diaphragm and hindlimb counterparts under differentiation pressure. In neuromuscular co-culture assays, AAV-delivery of Cxcl12 to G93A-hindlimb SC-derived myotubes enhances motor neuron axon extension and innervation, recapitulating the innervation capacity of EOM SC-derived myotubes. G93A mice fed with sodium butyrate (NaBu) supplementation exhibited less NMJ loss in hindlimb and diaphragm muscles. Additionally, SCs derived from G93A hindlimb and diaphragm muscles displayed elevated expression of Cxcl12 and improved renewability following NaBu treatment in vitro . Thus, the NaBu-induced transcriptomic changes resembling the patterns of EOM SCs may contribute to the beneficial effects observed in G93A mice. More broadly, the distinct transcriptomic profile of EOM SCs may offer novel therapeutic targets to slow progressive neuromuscular functional decay in ALS and provide possible "response biomarkers" in pre-clinical and clinical studies.

Quantification of autophagy flux in isolated mouse skeletal muscle fibers with overexpression of fluorescent protein mCherry-EGFP-LC3.

Evaluation of autophagy flux could be challenging for muscle fibers due to the baseline expression of mCherry-EGFP-LC3 along the Z-line. We established a protocol to overcome this difficulty. We overexpress mChery-EGFP-LC3 in the FDB muscle of an adult mouse via electroporation. Then, we enzymatically digest FDB muscle to yield individual fibers for live cell imaging. Finally, we develop an ImageJ-based program to eliminate the baseline striation pattern and semi-automatically quantify autophagosomes (APs) and autolysosomes (ALs) for autophagy flux analysis.

Deficient Sarcolemma Repair in ALS: A Novel Mechanism with Therapeutic Potential.

The plasma membrane (sarcolemma) of skeletal muscle myofibers is susceptible to injury caused by physical and chemical stresses during normal daily movement and/or under disease conditions. These acute plasma membrane disruptions are normally compensated by an intrinsic membrane resealing process involving interactions of multiple intracellular proteins including dysferlin, annexin, caveolin, and Mitsugumin 53 (MG53)/TRIM72. There is new evidence for compromised muscle sarcolemma repair mechanisms in Amyotrophic Lateral Sclerosis (ALS). Mitochondrial dysfunction in proximity to neuromuscular junctions (NMJs) increases oxidative stress, triggering MG53 aggregation and loss of its function. Compromised membrane repair further worsens sarcolemma fragility and amplifies oxidative stress in a vicious cycle. This article is to review existing literature supporting the concept that ALS is a disease of oxidative-stress induced disruption of muscle membrane repair that compromise the integrity of the NMJs and hence augmenting muscle membrane repair mechanisms could represent a viable therapeutic strategy for ALS.

Butyrate Ameliorates Mitochondrial Respiratory Capacity of The Motor-Neuron-like Cell Line NSC34-G93A, a Cellular Model for ALS.

Mitochondrial defects in motor neurons are pathological hallmarks of ALS, a neuromuscular disease with no effective treatment. Studies have shown that butyrate, a natural gut-bacteria product, alleviates the disease progression of ALS mice overexpressing a human ALS-associated mutation, hSOD1G93A. In the current study, we examined the potential molecular mechanisms underlying the effect of butyrate on mitochondrial function in cultured motor-neuron-like NSC34 with overexpression of hSOD1G93A (NSC34-G93A). The live cell confocal imaging study demonstrated that 1mM butyrate in the culture medium improved the mitochondrial network with reduced fragmentation in NSC34-G93A cells. Seahorse analysis revealed that NSC34-G93A cells treated with butyrate showed an increase of ~5-fold in mitochondrial Spare Respiratory Capacity with elevated Maximal Respiration. The time-dependent changes in the mRNA level of PGC1α, a master regulator of mitochondrial biogenesis, revealed a burst induction with an early increase (~5-fold) at 4 h, a peak at 24 h (~19-fold), and maintenance at 48 h (8-fold) post-treatment. In line with the transcriptional induction of PGC1α, both the mRNA and protein levels of the key molecules (MTCO1, MTCO2, and COX4) related to the mitochondrial electron transport chain were increased following the butyrate treatment. Our data indicate that activation of the PGC1α signaling axis could be one of the molecular mechanisms underlying the beneficial effects of butyrate treatment in improving mitochondrial bioenergetics in NSC34-G93A cells.

MG53 Preserves Neuromuscular Junction Integrity and Alleviates ALS Disease Progression.

Respiratory failure from progressive respiratory muscle weakness is the most common cause of death in amyotrophic lateral sclerosis (ALS). Defects in neuromuscular junctions (NMJs) and progressive NMJ loss occur at early stages, thus stabilizing and preserving NMJs represents a potential therapeutic strategy to slow ALS disease progression. Here we demonstrate that NMJ damage is repaired by MG53, an intrinsic muscle protein involved in plasma membrane repair. Compromised diaphragm muscle membrane repair and NMJ integrity are early pathological events in ALS. Diaphragm muscles from ALS mouse models show increased susceptibility to injury and intracellular MG53 aggregation, which is also a hallmark of human muscle samples from ALS patients. We show that systemic administration of recombinant human MG53 protein in ALS mice protects against injury to diaphragm muscle, preserves NMJ integrity, and slows ALS disease progression. As MG53 is present in circulation in rodents and humans under physiological conditions, our findings provide proof-of-concept data supporting MG53 as a potentially safe and effective therapy to mitigate ALS progression.

Butyrate Feeding Reverses CypD-Related Mitoflash Phenotypes in Mouse Myofibers.

Mitoflashes are spontaneous transients of the biosensor mt-cpYFP. In cardiomyocytes, mitoflashes are associated with the cyclophilin D (CypD) mediated opening of mitochondrial permeability transition pore (mPTP), while in skeletal muscle they are considered hallmarks of mitochondrial respiration burst under physiological conditions. Here, we evaluated the potential association between mitoflashes and the mPTP opening at different CypD levels and phosphorylation status by generating three CypD derived fusion constructs with a red shifted, pH stable Ca2+ sensor jRCaMP1b. We observed perinuclear mitochondrial Ca2+ efflux accompanying mitoflashes in CypD and CypDS42A (a phosphor-resistant mutation at Serine 42) overexpressed myofibers but not the control myofibers expressing the mitochondria-targeting sequence of CypD (CypDN30). Assisted by a newly developed analysis program, we identified shorter, more frequent mitoflash activities occurring over larger areas in CypD and CypDS42A overexpressed myofibers than the control CypDN30 myofibers. These observations provide an association between the elevated CypD expression and increased mitoflash activities in hindlimb muscles in an amyotrophic lateral sclerosis (ALS) mouse model previously observed. More importantly, feeding the mice with sodium butyrate reversed the CypD-associated mitoflash phenotypes and protected against ectopic upregulation of CypD, unveiling a novel molecular mechanism underlying butyrate mediated alleviation of ALS progression in the mouse model.

Physiological Ca2+ Transients Versus Pathological Steady-State Ca2+ Elevation, Who Flips the ROS Coin in Skeletal Muscle Mitochondria.

Mitochondria are both the primary provider of ATP and the pivotal regulator of cell death, which are essential for physiological muscle activities. Ca2+ plays a multifaceted role in mitochondrial function. During muscle contraction, Ca2+ influx into mitochondria activates multiple enzymes related to tricarboxylic acid (TCA) cycle and oxidative phosphorylation, resulting in increased ATP synthesis to meet the energy demand. Pathophysiological conditions such as skeletal muscle denervation or unloading also lead to elevated Ca2+ levels inside mitochondria. However, the outcomes of this steady-state elevation of mitochondrial Ca2+ level include exacerbated reactive oxygen species (ROS) generation, sensitized opening of mitochondrial permeability transition pore (mPTP), induction of programmed cell death, and ultimately muscle atrophy. Previously, both acute and long-term endurance exercises have been reported to activate certain signaling pathways to counteract ROS production. Meanwhile, electrical stimulation is known to help prevent apoptosis and alleviate muscle atrophy in denervated animal models and patients with motor impairment. There are various mechanistic studies that focus on the excitation-transcription coupling framework to understand the beneficial role of exercise and electrical stimulation. Interestingly, a recent study has revealed an unexpected role of rapid mitochondrial Ca2+ transients in keeping mPTP at a closed state with reduced mitochondrial ROS production. This discovery motivated us to contribute this review article to inspire further discussion about the potential mechanisms underlying differential outcomes of physiological mitochondrial Ca2+ transients and pathological mitochondrial Ca2+ elevation in skeletal muscle ROS production.

Dysregulated mitochondrial Ca2+ and ROS signaling in skeletal muscle of ALS mouse model.

Amyotrophic lateral sclerosis (ALS) is a devastating neuromuscular disease characterized by motor neuron loss and prominent skeletal muscle wasting. Despite more than one hundred years of research efforts, the pathogenic mechanisms underlying neuromuscular degeneration in ALS remain elusive. While the death of motor neuron is a defining hallmark of ALS, accumulated evidences suggested that in addition to being a victim of motor neuron axonal withdrawal, the intrinsic skeletal muscle degeneration may also actively contribute to ALS disease pathogenesis and progression. Examination of spinal cord and muscle autopsy/biopsy samples of ALS patients revealed similar mitochondrial abnormalities in morphology, quantity and disposition, which are accompanied by defective mitochondrial respiratory chain complex and elevated oxidative stress. Detailing the molecular/cellular mechanisms and the role of mitochondrial dysfunction in ALS relies on ALS animal model studies. This review article discusses the dysregulated mitochondrial Ca2+ and reactive oxygen species (ROS) signaling revealed in live skeletal muscle derived from ALS mouse models, and a potential role of the vicious cycle formed between the dysregulated mitochondrial Ca2+ signaling and excessive ROS production in promoting muscle wasting during ALS progression.

ROS-related mitochondrial dysfunction in skeletal muscle of an ALS mouse model during the disease progression.

In amyotrophic lateral sclerosis (ALS), mitochondrial dysfunction and oxidative stress form a vicious cycle that promotes neurodegeneration and muscle wasting. To quantify the disease-stage-dependent changes of mitochondrial function and their relationship to the generation of reactive oxygen species (ROS), we generated double transgenic mice (G93A/cpYFP) that carry human ALS mutation SOD1G93A and mt-cpYFP transgenes, in which mt-cpYFP detects dynamic changes of ROS-related mitoflash events at individual mitochondria level. Compared with wild type mice, mitoflash activity in the SOD1G93A (G93A) mouse muscle showed an increased flashing frequency prior to the onset of ALS symptom (at the age of 2 months), whereas the onset of ALS symptoms (at the age of 4 months) is associated with drastic changes in the kinetics property of mitoflash signal with prolonged full duration at half maximum (FDHM). Elevated levels of cytosolic ROS in skeletal muscle derived from the SOD1G93A mice were confirmed with fluorescent probes, MitoSOX™ Red and ROS Brite™570. Immunoblotting analysis of subcellular mitochondrial fractionation of G93A muscle revealed an increased expression level of cyclophilin D (CypD), a regulatory component of the mitochondrial permeability transition pore (mPTP), at the age of 4 months but not at the age of 2 months. Transient overexpressing of SOD1G93A in skeletal muscle of wild type mice directly promoted mitochondrial ROS production with an enhanced mitoflash activity in the absence of motor neuron axonal withdrawal. Remarkably, the SOD1G93A-induced mitoflash activity was attenuated by the application of cyclosporine A (CsA), an inhibitor of CypD. Similar to the observation with the SOD1G93A transgenic mice, an increased expression level of CypD was also detected in skeletal muscle following transient overexpression of SOD1G93A. Overall, this study reveals a disease-stage-dependent change in mitochondrial function that is associated with CypD-dependent mPTP opening; and the ALS mutation SOD1G93A directly contributes to mitochondrial dysfunction in the absence of motor neuron axonal withdrawal.

Irisin Protects Heart Against Ischemia-Reperfusion Injury Through a SOD2-Dependent Mitochondria Mechanism.

Irisin, a muscle-origin protein derived from the extracellular domain of the fibronectin domain-containing 5 protein (FNDC5), has been shown to modulate mitochondria welfare through paracrine action. Here, we test the hypothesis that irisin contributes to cardioprotection after myocardial infarction by preserving mitochondrial function in cardiomyocytes. Animal model studies show that intravenous administration of exogenous irisin produces dose-dependent protection against ischemia/reperfusion (I/R)-induced injury to the heart as reflected by the improvement of left ventricular ejection fraction and the reduction in serum level of cTnI (n = 15, P < 0.05). I/R-induced apoptosis of cardiomyocytes is reduced after irisin treatment. The irisin-mediated protection has, at least in part, an effect on mitochondrial function because administration of irisin increases irisin staining in the mitochondria of the infarct area. Irisin also reduces I/R-induced oxidative stress as determined by mitochondrial membrane potential evaluation and superoxide FLASH event recording (n = 4, P < 0.05). The interaction between irisin and superoxide dismutase2 (SOD2) plays a key role in the protective process because irisin treatment increases SOD activity (n = 10, P < 0.05) and restores the mitochondria localization of SOD2 in cardiomyocytes (n = 5, P < 0.05). These results demonstrate that irisin plays a protective role against I/R injury to the heart. Targeting the action of irisin in mitochondria presents a novel therapeutic intervention for myocardial infarction.

ß-aminoisobutyric Acid, l-BAIBA, Is a Muscle-Derived Osteocyte Survival Factor.

Exercise has beneficial effects on metabolism and on tissues. The exercise-induced muscle factor ß-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.

ALS-associated mutation SOD1G93A leads to abnormal mitochondrial dynamics in osteocytes.

While the death of motor neuron is a pathological hallmark of amyotrophic lateral sclerosis (ALS), defects in other cell types or organs may also actively contribute to ALS disease progression. ALS patients experience progressive skeletal muscle wasting that may not only exacerbate neuronal degeneration, but likely has a significant impact on bone function. In our previous published study, we have discovered severe bone loss in an ALS mouse model with overexpression of ALS-associated mutation SOD1G93A (G93A). Here we further provide a mechanistic understanding of the bone loss in ALS animal and cellular models. Combining mitochondrial fluorescent indicators and confocal live cell imaging, we discovered abnormalities in mitochondrial network and dynamics in primary osteocytes derived from the same ALS mouse model G93A. Those mitochondrial defects occur in ALS mice after the onset of neuromuscular symptoms, indicating that mitochondria in bone cells respond to muscle atrophy during ALS disease progression. To examine whether ALS mutation has a direct contribution to mitochondrial dysfunction independent of muscle atrophy, we evaluated mitochondrial morphology and motility in cultured osteocytes (MLO-Y4) with overexpression of mitochondrial targeted SOD1G93A. Compared with osteocytes overexpressing the wild type SOD1 as a control, the SOD1G93A osteocytes showed similar defects in mitochondrial network and dynamic as that of the primary osteocytes derived from the ALS mouse model. In addition, we further discovered that overexpression of SOD1G93A enhanced the expression level of dynamin-related protein 1 (Drp1), a key protein promoting mitochondrial fission activity, and reduced the expression level of optic atrophy protein 1 (OPA1), a key protein related to mitochondrial fusion. A specific mitochondrial fission inhibitor (Mdivi-1) partially reversed the effect of SOD1G93A on mitochondrial network and dynamics, indicating that SOD1G93A likely promotes mitochondrial fission, but suppresses the fusion activity. Our data provide the first evidence that mitochondria show abnormality in osteocytes derived from an ALS mouse model. The accumulation of mutant SOD1G93A protein inside mitochondria directly causes dysfunction in mitochondrial dynamics in cultured MLO-Y4 osteocytes. In addition, the ALS mutation SOD1G93A-mediated dysfunction in mitochondrial dynamics is associated with an enhanced apoptosis in osteocytes, which could be a potential mechanism underlying the bone loss during ALS progression.

Irisin protects mitochondria function during pulmonary ischemia/reperfusion injury.

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.

Absence of physiological Ca2+ transients is an initial trigger for mitochondrial dysfunction in skeletal muscle following denervation.

BACKGROUND: Motor neurons control muscle contraction by initiating action potentials in muscle. Denervation of muscle from motor neurons leads to muscle atrophy, which is linked to mitochondrial dysfunction. It is known that denervation promotes mitochondrial reactive oxygen species (ROS) production in muscle, whereas the initial cause of mitochondrial ROS production in denervated muscle remains elusive. Since denervation isolates muscle from motor neurons and deprives it from any electric stimulation, no action potentials are initiated, and therefore, no physiological Ca2+ transients are generated inside denervated muscle fibers. We tested whether loss of physiological Ca2+ transients is an initial cause leading to mitochondrial dysfunction in denervated skeletal muscle. METHODS: A transgenic mouse model expressing a mitochondrial targeted biosensor (mt-cpYFP) allowed a real-time measurement of the ROS-related mitochondrial metabolic function following denervation, termed "mitoflash." Using live cell imaging, electrophysiological, pharmacological, and biochemical studies, we examined a potential molecular mechanism that initiates ROS-related mitochondrial dysfunction following denervation. RESULTS: We found that muscle fibers showed a fourfold increase in mitoflash activity 24 h after denervation. The denervation-induced mitoflash activity was likely associated with an increased activity of mitochondrial permeability transition pore (mPTP), as the mitoflash activity was attenuated by application of cyclosporine A. Electrical stimulation rapidly reduced mitoflash activity in both sham and denervated muscle fibers. We further demonstrated that the Ca2+ level inside mitochondria follows the time course of the cytosolic Ca2+ transient and that inhibition of mitochondrial Ca2+ uptake by Ru360 blocks the effect of electric stimulation on mitoflash activity. CONCLUSIONS: The loss of cytosolic Ca2+ transients due to denervation results in the downstream absence of mitochondrial Ca2+ uptake. Our studies suggest that this could be an initial trigger for enhanced mPTP-related mitochondrial ROS generation in skeletal muscle.

Target Intestinal Microbiota to Alleviate Disease Progression in Amyotrophic Lateral Sclerosis.

PURPOSE: Emerging evidence has demonstrated that gut microbiome plays essential roles in the pathogenesis of human diseases in distal organs. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. Treatment with the only drug approved by the US Food and Drug Administration for use in ALS, riluzole, extends a patient׳s life span by only a few months. Thus, there is an urgent need to develop novel interventions that for alleviate disease progression and improve quality of life in patients with ALS. 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 before ALS disease onset. The mice were given 2% butyrate, a natural bacterial product, in the drinking water. RESULTS: In mice fed with butyrate, intestinal microbial homeostasis was restored, gut integrity was improved, and life span was prolonged compared with those in control mice. At the cellular level, abnormal Paneth cells-specialized intestinal epithelial cells that regulate the host-bacterial interactions-were significantly decreased in the ALS mice treated with butyrate. In both ALS mice and intestinal epithelial cells cultured from humans, butyrate treatment was associated with decreased aggregation of the G93A superoxide dismutase 1 mutated protein. IMPLICATIONS: The findings from this study highlight the complex role of the gut microbiome and intestinal epithelium in the progression of ALS and present butyrate as a potential therapeutic reagent for restoring ALS-related dysbiosis.

Mitochondrial Ca(2+) uptake in skeletal muscle health and disease.

Muscle uses Ca(2+) as a messenger to control contraction and relies on ATP to maintain the intracellular Ca(2+) homeostasis. Mitochondria are the major sub-cellular organelle of ATP production. With a negative inner membrane potential, mitochondria take up Ca(2+) from their surroundings, a process called mitochondrial Ca(2+) uptake. Under physiological conditions, Ca(2+) uptake into mitochondria promotes ATP production. Excessive uptake causes mitochondrial Ca(2+) overload, which activates downstream adverse responses leading to cell dysfunction. Moreover, mitochondrial Ca(2+) uptake could shape spatio-temporal patterns of intracellular Ca(2+) signaling. Malfunction of mitochondrial Ca(2+) uptake is implicated in muscle degeneration. Unlike non-excitable cells, mitochondria in muscle cells experience dramatic changes of intracellular Ca(2+) levels. Besides the sudden elevation of Ca(2+) level induced by action potentials, Ca(2+) transients in muscle cells can be as short as a few milliseconds during a single twitch or as long as minutes during tetanic contraction, which raises the question whether mitochondrial Ca(2+) uptake is fast and big enough to shape intracellular Ca(2+) signaling during excitation-contraction coupling and creates technical challenges for quantification of the dynamic changes of Ca(2+) inside mitochondria. This review focuses on characterization of mitochondrial Ca(2+) uptake in skeletal muscle and its role in muscle physiology and diseases.

Muscle-Bone Crosstalk in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS), also called Lou Gehrig's disease, is a fatal neuromuscular disorder characterized by degeneration of motor neurons and by skeletal muscle atrophy. Although the death of motor neurons is a pathological hallmark of ALS, the potential role of other organs in disease progression remains to be elucidated. Skeletal muscle and bone are the two largest organs in the human body. They are responsible not only for locomotion but also for maintaining whole body normal metabolism and homeostasis. Patients with ALS display severe muscle atrophy, which may reflect intrinsic defects in mitochondrial respiratory function and calcium (Ca) signaling in muscle fibers, in addition to the role of axonal withdrawal associated with ALS progression. Incidence of fractures is high in ALS patients, indicating there are potential bone defects in individuals with this condition. There is a lifelong interaction between skeletal muscle and bone. The severe muscle degeneration that occurs during ALS progression may potentially have a significant impact on bone function, and the defective bone may also contribute significantly to neuromuscular degeneration in the course of the disease. Due to the nature of the rapid and severe neuromuscular symptoms, a majority of studies on ALS have focused on neurodegeneration. Just a few studies have explored the possible contribution of muscle defects, even fewer on bone defects, and fewer still on possible muscle-bone crosstalk in ALS. This review article discusses current studies on bone defects and potential defects in muscle-bone crosstalk in ALS.

Leaky intestine and impaired microbiome in an amyotrophic lateral sclerosis mouse model.

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 (SOD1(G93A)), 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.

Impaired bone homeostasis in amyotrophic lateral sclerosis mice with muscle atrophy.

There is an intimate relationship between muscle and bone throughout life. However, how alterations in muscle functions in disease impact bone homeostasis is poorly understood. Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by progressive muscle atrophy. In this study we analyzed the effects of ALS on bone using the well established G93A transgenic mouse model, which harbors an ALS-causing mutation in the gene encoding superoxide dismutase 1. We found that 4-month-old G93A mice with severe muscle atrophy had dramatically reduced trabecular and cortical bone mass compared with their sex-matched wild type (WT) control littermates. Mechanically, we found that multiple osteoblast properties, such as the formation of osteoprogenitors, activation of Akt and Erk1/2 pathways, and osteoblast differentiation capacity, were severely impaired in primary cultures and bones from G93A relative to WT mice; this could contribute to reduced bone formation in the mutant mice. Conversely, osteoclast formation and bone resorption were strikingly enhanced in primary bone marrow cultures and bones of G93A mice compared with WT mice. Furthermore, sclerostin and RANKL expression in osteocytes embedded in the bone matrix were greatly up-regulated, and ß-catenin was down-regulated in osteoblasts from G93A mice when compared with those of WT mice. Interestingly, calvarial bone that does not load and long bones from 2-month-old G93A mice without muscle atrophy displayed no detectable changes in parameters for osteoblast and osteoclast functions. Thus, for the first time to our knowledge, we have demonstrated that ALS causes abnormal bone remodeling and defined the underlying molecular and cellular mechanisms.