Pharmacology/Neurobiology (Rüegg)Head of Research Unit Prof. Dr.Markus A. RüeggOverviewMembersPublicationsProjects & CollaborationsProjects & Collaborations OverviewMembersPublicationsProjects & Collaborations Projects & Collaborations 36 foundShow per page10 10 20 50 Deciphering the molecular basis of selective muscle vulnerability in Myasthenia Gravis Research Project | 1 Project MembersImported from Grants Tool 4702817 Characterization of pathophysiological mechanisms involved in the selective vulnerability of muscles in Myasthenia Gravis Research Project | 2 Project MembersNo Description available Locations, interactions, functions and expression (LIFE) of signals involved in sarcopenia Research Project | 1 Project MembersNo Description available Sinergia: Machine-learned Design and Bioxolography of Functional 3D Skeletal Muscle Tissues Research Project | 1 Project MembersEngineered 3D skeletal muscle tissue (SMT) is an important tool to study muscle physiology and disease. SMT engineering has applications in regenerative medicine, in vitro drug screening, bio-hybrid robotics, and cultured meat. However, state-of-the-art engineered muscle tissue does not effectively mimic the cellular heterogeneity, architecture, and performance of biological muscle. To address this, researchers are developing techniques to bioprint, differentiate, and mature functional muscle architectures. Machine Learning (ML) approaches could streamline SMT design by rapidly exploring the ideal conditions for muscle biofabrication, computationally capturing the complex interplay between biofabrication parameters (bioprinting, differentiation, and mechanical and electrical maturation), and the resulting functionality of the contractile SMT. This project consists of four key objectives: (1) Adaptation of xolography1 to muscle bioprinting. We will develop the bioxolography technique to fabricate highly aligned and anisotropic cell-laden hydrogels without constraints on the achievable shapes. (2) Biofabrication and differentiation of 3D heterocellular muscle constructs. We will develop a protocol to bioprint, culture, and differentiate arrays of muscle bundles which contain multiple cell types, thereby mimicking the natural muscle. (3) Maturation and characterization of engineered muscle tissues. We will mechanically stimulate our engineered muscle tissues for maturation and characterize their cellular morphology and contractile performance. (4) Development of an ML pipeline to guide the biofabrication and design of muscle actuators. Finally, we will develop an ML pipeline which maps biofabrication and tissue engineering parameters to performance metrics of engineered muscle (structure and function), leveraging a differentiable simulation approach originally developed to model soft material and actuator deformation in soft robotics. We will demonstrate proof-of-concept of this model by designing a centimeter-scale asymmetric, antagonistically actuated skeletal muscle construct. Methods. (1) We will print highly aligned, multinucleated and large (2 to 3-cm-long) bundles of muscle fibers by developing bioxolography: a linear volumetric bioprinting process (LVBP), realized by designing new photoresins and photoinitiators for previously established xolographic printing. (2) Primary murine myoblasts will be co-printed with fibro-adipogenic progenitors (FAPs), and cell culture will be optimized to promote myogenesis, myogenic cell differentiation, and tissue maturation in three dimensional tissue. (3) Muscle tissue will be mechanically stimulated under varying conditions, and characterized for structure and response to electrical stimulation. In a further iteration, we will also characterize muscle tissue co-cultured with optogenetically modified motor neurons to realize a light-controllable innervation system for remote neural actuation of muscle tissue. (4) We will create an in-silico-to-in-vitro platform to optimize muscle design by using ML and our differentiable finite element method (FEM) to map biofabrication parameters to SMT's contractility. This project will accelerate and improve the design and fabrication of functional SMTs by providing an in-silico-to-in-vitro platform for 3D muscle construct design. Our work will provide insights into biofabrication, soft materials, 3D cell culture techniques, hetero-cellular models, bio-hybrid technologies, robotic design, and biophysical cell stimulation. We anticipate multidisciplinary advancements in tissue engineering, muscle physiopathology, 3D bioprinting, the development of new therapeutics, biological machine learning, bio-hybrid robotics, and engineering with living materials. Role of Fam134b-driven endoplasmic reticulum (ER)-phagy in sarcopenia Research Project | 1 Project MembersAutophagy is a process by which cells remove damaged organelles or protein aggregates to maintain cellular homeostasis. ER-phagy is a recently identified form of autophagy, which clears damaged parts of ER membrane. To elicit ER-phagy, specific ER-bound proteins recognize ER damage and recruit autophagy machinery. The ER is particularly interesting in myofibers because it adopts a unique structure of a very thin sheet attached underneath the plasma membrane. Proper ion flux through muscle ER is essential for contraction. Also, ER is an integral component of anchoring myonuclei at the cell periphery. Despite the obvious importance of the ER in myofibers, it is unknown how the quality of the ER is controlled and whether it is affected during ageing. From snRNA-Seq studies, a novel nuclear population was identified in aged muscles that included Fam134b as a distinguishing marker gene. Our consortium independently confirmed that Fam134b transcripts are increased during muscle ageing in bulk RNA-seq (Börsch et al., 2021; Fig. 1B) and snRNA-FISH (Fig. 1C). We hypothesize that increased Fam134b expression drives ER-phagy during muscle ageing and, importantly, that manipulation of ER-phagy can mitigate sarcopenia. To begin to tackle this question, we have joined forces between two groups with expertise on myonuclear gene expression (Kim) and muscle ageing and mTORC1 signaling (Rüegg), respectively. With the support of Eucor_seed, we aim to obtain foundational data to strengthen our hypothesis and secure larger future funding. Specifically, we will 1) test our core idea that ER-phagy flux is altered in aged muscles and examine its relationship to the mTORC1 pathway, 2) investigate how modulating Fam134b expression affects muscle performance in young and old mice, and 3) take the first step in identifying upstream regulators of Fam134b expression during ageing. Spatial transcriptomics: Novel methods to detect pre-symptomatic causes of Sarcopenia Research Project | 2 Project MembersAs overall life expectancy increases so do comorbidities associated with an ageing population. Sarcopenia, the progressive loss of skeletal muscle mass and function, is widespread amongst the elderly and leads to reduced quality of life, frailty, and ultimately death - representing a major public health issue and clinical challenge. Sarcopenia entails complex pathogenesis involving neuromuscular function, and metabolism changes are suggested to be the first sign of muscle ageing. Thus, understanding these could help detect pre-symptomatic sarcopenia. The multinucleated composition of myofibers has hampered single-cell analysis, but efforts in bulk, single-nuclear RNA-seq and multiplex-FISH identified sarcopenic changes originating from differences in cell populations [1]. Therefore, the only methodology able to pinpoint the sub-cellular and cell-specific localisation of signals driving sarcopenia is spatial transcriptomics [2-3]. Most workflows however have limited gene coverage, and some proprietary methods with higher capability seem not to be fully reproducible. Therefore, the research community requires openly accessible protocols allowing the resolution of >10,000 genes at sub-diffraction-limit which facilitates distinction of transcripts in immediate proximity, to further encourage innovation in sarcopenic research and ultimately healthcare. This project aims to address this and will create methods, including detailed instructions on probe design, available for others to reproduce. Linker-based gene therapy of LAMA2-related muscular dystrophy using AAV-MYO Research Project | 1 Project MembersExtracellular matrix (ECM) acts as scaffold to provide essential structural support to the surrounding cells. Laminins are essential components of the ECM connecting it tightly with the underlying cell layer, just like the binds between scaffolding and a building. Mutations in this protein family cause a range of severe disorders that affect different organs. The rope-like laminin-211 protein, composed of three intertwined laminin chains, is the main isoform in skeletal muscle. Mutations in the LAMA2 gene, encoding one of the chains in laminin-211, leads to its loss and causes a rare, severe and early-onset muscular dystrophy, called MDC1A or LAMA2 MD. Affected children show muscle wasting and weakness and eventually die from respiratory insufficiency. There is no treatment for LAMA2 MD. We have shown that two specifically designed linker proteins strongly improve the pathology and lifespan in LAMA2 MD mice, which lack laminin-211 like LAMA2 MD patients and display very similar symptoms. In ongoing research, we are developing a gene therapy approach to deliver the two linkers to LAMA2 MD mice by using an adeno-associated viral vector (AAV9). Our initial results show that this gene therapy approach is indeed possible. We now aim to optimize linker delivery using newly developed AAV9 variants (myotropic AAVs) that are even more efficient at infecting skeletal muscles. We are hopeful these experiments will pave the way to a gene therapy treatment for LAMA2 MD patients. A multidimensional single-cell approach to understand muscle dystrophy Research Project | 1 Project MembersDuchenne Muscular Dystrophy (DMD) and LAMA2-related Muscular Dystrophy (LAMA2 MD) are devastating rare genetic diseases of childhood manifested by progressive skeletal muscle wasting and ultimately death. In both diseases, the muscle undergoes constant cycles of degeneration-regeneration exacerbated by intrinsic muscle stem cell (MuSC) dysfunction and their impaired ability to support long-term regeneration. Till date, the nature of the cellular changes that contribute to muscle pathologies are unknown. Specifically, there is critical lack of research that provides an unbiased elucidation of the cellular events underlying the different steps of muscle disease progression at the single-cell level. This impedes development of therapeutic strategies that can effectively treat these devastating diseases. Currently, there is no known cure for LAMA2 MD and no therapy that can halt DMD disease progression. The overall goal of our project is to use a combination of single-cell transcriptomics (RNA-sequencing) and proteomics (mass cytometry, CyTOF) to define the cellular composition of diseased muscle tissues at the single-cell resolution. We will delineate the different cell populations that pre-exist and arise during disease progression and classify the novel cellular subsets involved in this process. This data, in combination with genetic lineage tracing will allow reconstruction of the MuSC lineage hierarchy. We will further characterize cellular subpopulations associated with human muscle dystrophies by performing 3-Dimensional intact-tissue RNA sequencing on patient biopsies. Thus, MYOCITY will significantly advance the current understanding of muscle dystrophies, uncover disease-related subsets in human biopsies and also develop new biomarkers and eventually new pharmacological approaches that will stimulate intrinsic muscle tissue repair. Origin and function of signals driving sarcopenia and neuromuscular synapse destabilization Research Project | 1 Project MembersLead Verlust von Muskelmasse und Funktion im Alter führt zu Gangunsicherheiten und dadurch zu einer erhöhten Sturzgefahr. Die molekularen Ursachen dieses «Sarkopenie» genannten Muskelschwunds sind jedoch wenig erforscht. Dieses Projekt möchte einen Beitrag leisten, dessen Ursachen besser zu verstehen. Lay summary Bei einem normal gewichtigen Menschen liegt der Muskelanteil bei bis zu 50% der Körpermasse, kann jedoch durch viele äussere Einflüsse (Sport oder Immobilisierung) verändert werden. Gezieltes Muskelaufbautraining aktiviert einen Signalweg in den Muskelfasern, welcher die Proteinsynthese stimuliert. Ist der gleiche Signalweg jedoch in Muskelfasern von genetisch veränderten Mäusen ständig aktiv, nimmt die Muskelmasse ab und die Tiere entwickeln eine frühzeitige Sarkopenie mit Veränderungen in den gleichen Signalwegen wie bei alten Mäusen. In den geplanten Experimenten werden wir diese Signalwege funktionell untersuchen und die Zellen identifizieren, welche für die altersbedingten Veränderungen verantwortlich sind. Zudem werden wir die Auswirkungen des Alterns auf die Verbindung zwischen Nerven und Muskelfasern genauer untersuchen. Dieses Projekt trägt zu einem besseren Verständnis der Ursachen von Sarkopenie bei. Daraus können in Zukunft vielleicht Therapieansätze entwickelt werden, welche den Verlust an Muskelmasse im Alter verlangsamen oder gar verhindern. Solche Therapien haben das Potential die Lebensqualität der ständig älter werdenden Bevölkerung zu verbessern. The role of mTORC1 in muscle proteostasis Research Project | 2 Project MembersSkeletal muscle accounts for up to 50% of the entire body weight in humans. It is essential for locomotion and breathing and it affects whole-body metabolism. Preservation of muscle mass is thus critical to maintain body function and health. Current views indicate that muscle mass is controlled by the tight balance between protein synthesis and protein degradation, called proteostasis. Perturbation of this balance by extrinsic factors, as for example seen in cachexia (i.e., muscle loss as a secondary consequence of e.g. cancer, AIDS, or cardiac and kidney disease) or in sarcopenia (i.e., loss of muscle mass and function as a consequence of aging), is a main cause of loss of life quality and increased mortality. Thus, a better molecular understanding of muscle proteostasis is of fundamental importance to develop possible treatment strategies to counteract the above diseases. Proteostasis is thought to be controlled (1) by the mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) by regulating protein synthesis (Laplante and Sabatini, 2012) and (2) by forkhead box O (FoxO) transcription factors by regulating expression of the proteins involved in protein degradation (Milan et al., 2015; Zhao et al., 2007). Thus, inhibition of mTORC1 and/or activation of FoxO would cause muscle loss (atrophy), whereas activation of mTORC1 and/or inhibition of FoxO would result in muscle gain (hypertrophy). Consistent with this notion, genetic inactivation of mTORC1 by muscle-specific depletion of the mTORC1-essential component raptor causes muscle atrophy (Bentzinger et al., 2008). However and in striking contrast to the expected outcome of muscle gain, sustained activation of mTORC1 in muscle by knockout of its upstream inhibitor Tsc1 (TSCmKO mice) also results in atrophy, severe myopathy and early death (Bentzinger et al., 2013; Castets et al., 2013). This phenotype is observed despite the marked increase in protein synthesis. Hence, the mechanisms involved in muscle atrophy in TSCmKO mice remains unresolved. Marco Kaiser investigated the mechanisms that may underlie this phenotype during his Ph.D. thesis. In particular, he investigated the role of the most important protein degradation pathway of cells, which is the ubiquitin-proteasome system (UPS). His work has shown that sustained activation of mTORC1 in muscles of TSCmKO mice indeed leads to a significant increase in the expression of all the proteasomal subunits and increased UPS activity. Marco also observed an increase in the activity of the proteasome in lysates of TSCmKO muscles using an in vitro assay. Interestingly, the same increase in proteasome activity was observed in "inducible-TSCmKO" (iTSCmKO) mice upon acute muscle-specific deletion of Tsc1 for 3 weeks. This increase in proteasome activity was, at least in part, reversed by short-term treatment of the TSCmKO mice with the mTORC1-inhibitor rapamycin. Marco also discovered that increased UPS activity was accompanied by a concomitant increase of the transcription factor "nuclear factor, erythroid-derived 2,-like 1" (Nfe2l1, also called Nrf1). Moreover, he found evidence that Nrf1 may regulate the UPS in TSCmKO mice. We now aim to establish that this increase in UPS activity is the mechanism that causes muscle atrophy in TSCmKO mice and to test the functional role of Nrf1 in the regulation of the UPS. To do this, we will perturb the function of the UPS by bortezomib (BTZ) and by knocking down Nrf1 by shRNA in skeletal muscle in vivo. We hope that these experiments will firmly establish that mTORC1 activation is the main driver of protein degradation in skeletal muscle and that Nrf1 is the main regulator of this pathway. These experiments are important to better understand the control of muscle mass and to eventually develop new therapeutic agents that could slow-down the massive muscle wasting observed in cachexia and sarcopenia. 1234 1...4 OverviewMembersPublicationsProjects & Collaborations
Projects & Collaborations 36 foundShow per page10 10 20 50 Deciphering the molecular basis of selective muscle vulnerability in Myasthenia Gravis Research Project | 1 Project MembersImported from Grants Tool 4702817 Characterization of pathophysiological mechanisms involved in the selective vulnerability of muscles in Myasthenia Gravis Research Project | 2 Project MembersNo Description available Locations, interactions, functions and expression (LIFE) of signals involved in sarcopenia Research Project | 1 Project MembersNo Description available Sinergia: Machine-learned Design and Bioxolography of Functional 3D Skeletal Muscle Tissues Research Project | 1 Project MembersEngineered 3D skeletal muscle tissue (SMT) is an important tool to study muscle physiology and disease. SMT engineering has applications in regenerative medicine, in vitro drug screening, bio-hybrid robotics, and cultured meat. However, state-of-the-art engineered muscle tissue does not effectively mimic the cellular heterogeneity, architecture, and performance of biological muscle. To address this, researchers are developing techniques to bioprint, differentiate, and mature functional muscle architectures. Machine Learning (ML) approaches could streamline SMT design by rapidly exploring the ideal conditions for muscle biofabrication, computationally capturing the complex interplay between biofabrication parameters (bioprinting, differentiation, and mechanical and electrical maturation), and the resulting functionality of the contractile SMT. This project consists of four key objectives: (1) Adaptation of xolography1 to muscle bioprinting. We will develop the bioxolography technique to fabricate highly aligned and anisotropic cell-laden hydrogels without constraints on the achievable shapes. (2) Biofabrication and differentiation of 3D heterocellular muscle constructs. We will develop a protocol to bioprint, culture, and differentiate arrays of muscle bundles which contain multiple cell types, thereby mimicking the natural muscle. (3) Maturation and characterization of engineered muscle tissues. We will mechanically stimulate our engineered muscle tissues for maturation and characterize their cellular morphology and contractile performance. (4) Development of an ML pipeline to guide the biofabrication and design of muscle actuators. Finally, we will develop an ML pipeline which maps biofabrication and tissue engineering parameters to performance metrics of engineered muscle (structure and function), leveraging a differentiable simulation approach originally developed to model soft material and actuator deformation in soft robotics. We will demonstrate proof-of-concept of this model by designing a centimeter-scale asymmetric, antagonistically actuated skeletal muscle construct. Methods. (1) We will print highly aligned, multinucleated and large (2 to 3-cm-long) bundles of muscle fibers by developing bioxolography: a linear volumetric bioprinting process (LVBP), realized by designing new photoresins and photoinitiators for previously established xolographic printing. (2) Primary murine myoblasts will be co-printed with fibro-adipogenic progenitors (FAPs), and cell culture will be optimized to promote myogenesis, myogenic cell differentiation, and tissue maturation in three dimensional tissue. (3) Muscle tissue will be mechanically stimulated under varying conditions, and characterized for structure and response to electrical stimulation. In a further iteration, we will also characterize muscle tissue co-cultured with optogenetically modified motor neurons to realize a light-controllable innervation system for remote neural actuation of muscle tissue. (4) We will create an in-silico-to-in-vitro platform to optimize muscle design by using ML and our differentiable finite element method (FEM) to map biofabrication parameters to SMT's contractility. This project will accelerate and improve the design and fabrication of functional SMTs by providing an in-silico-to-in-vitro platform for 3D muscle construct design. Our work will provide insights into biofabrication, soft materials, 3D cell culture techniques, hetero-cellular models, bio-hybrid technologies, robotic design, and biophysical cell stimulation. We anticipate multidisciplinary advancements in tissue engineering, muscle physiopathology, 3D bioprinting, the development of new therapeutics, biological machine learning, bio-hybrid robotics, and engineering with living materials. Role of Fam134b-driven endoplasmic reticulum (ER)-phagy in sarcopenia Research Project | 1 Project MembersAutophagy is a process by which cells remove damaged organelles or protein aggregates to maintain cellular homeostasis. ER-phagy is a recently identified form of autophagy, which clears damaged parts of ER membrane. To elicit ER-phagy, specific ER-bound proteins recognize ER damage and recruit autophagy machinery. The ER is particularly interesting in myofibers because it adopts a unique structure of a very thin sheet attached underneath the plasma membrane. Proper ion flux through muscle ER is essential for contraction. Also, ER is an integral component of anchoring myonuclei at the cell periphery. Despite the obvious importance of the ER in myofibers, it is unknown how the quality of the ER is controlled and whether it is affected during ageing. From snRNA-Seq studies, a novel nuclear population was identified in aged muscles that included Fam134b as a distinguishing marker gene. Our consortium independently confirmed that Fam134b transcripts are increased during muscle ageing in bulk RNA-seq (Börsch et al., 2021; Fig. 1B) and snRNA-FISH (Fig. 1C). We hypothesize that increased Fam134b expression drives ER-phagy during muscle ageing and, importantly, that manipulation of ER-phagy can mitigate sarcopenia. To begin to tackle this question, we have joined forces between two groups with expertise on myonuclear gene expression (Kim) and muscle ageing and mTORC1 signaling (Rüegg), respectively. With the support of Eucor_seed, we aim to obtain foundational data to strengthen our hypothesis and secure larger future funding. Specifically, we will 1) test our core idea that ER-phagy flux is altered in aged muscles and examine its relationship to the mTORC1 pathway, 2) investigate how modulating Fam134b expression affects muscle performance in young and old mice, and 3) take the first step in identifying upstream regulators of Fam134b expression during ageing. Spatial transcriptomics: Novel methods to detect pre-symptomatic causes of Sarcopenia Research Project | 2 Project MembersAs overall life expectancy increases so do comorbidities associated with an ageing population. Sarcopenia, the progressive loss of skeletal muscle mass and function, is widespread amongst the elderly and leads to reduced quality of life, frailty, and ultimately death - representing a major public health issue and clinical challenge. Sarcopenia entails complex pathogenesis involving neuromuscular function, and metabolism changes are suggested to be the first sign of muscle ageing. Thus, understanding these could help detect pre-symptomatic sarcopenia. The multinucleated composition of myofibers has hampered single-cell analysis, but efforts in bulk, single-nuclear RNA-seq and multiplex-FISH identified sarcopenic changes originating from differences in cell populations [1]. Therefore, the only methodology able to pinpoint the sub-cellular and cell-specific localisation of signals driving sarcopenia is spatial transcriptomics [2-3]. Most workflows however have limited gene coverage, and some proprietary methods with higher capability seem not to be fully reproducible. Therefore, the research community requires openly accessible protocols allowing the resolution of >10,000 genes at sub-diffraction-limit which facilitates distinction of transcripts in immediate proximity, to further encourage innovation in sarcopenic research and ultimately healthcare. This project aims to address this and will create methods, including detailed instructions on probe design, available for others to reproduce. Linker-based gene therapy of LAMA2-related muscular dystrophy using AAV-MYO Research Project | 1 Project MembersExtracellular matrix (ECM) acts as scaffold to provide essential structural support to the surrounding cells. Laminins are essential components of the ECM connecting it tightly with the underlying cell layer, just like the binds between scaffolding and a building. Mutations in this protein family cause a range of severe disorders that affect different organs. The rope-like laminin-211 protein, composed of three intertwined laminin chains, is the main isoform in skeletal muscle. Mutations in the LAMA2 gene, encoding one of the chains in laminin-211, leads to its loss and causes a rare, severe and early-onset muscular dystrophy, called MDC1A or LAMA2 MD. Affected children show muscle wasting and weakness and eventually die from respiratory insufficiency. There is no treatment for LAMA2 MD. We have shown that two specifically designed linker proteins strongly improve the pathology and lifespan in LAMA2 MD mice, which lack laminin-211 like LAMA2 MD patients and display very similar symptoms. In ongoing research, we are developing a gene therapy approach to deliver the two linkers to LAMA2 MD mice by using an adeno-associated viral vector (AAV9). Our initial results show that this gene therapy approach is indeed possible. We now aim to optimize linker delivery using newly developed AAV9 variants (myotropic AAVs) that are even more efficient at infecting skeletal muscles. We are hopeful these experiments will pave the way to a gene therapy treatment for LAMA2 MD patients. A multidimensional single-cell approach to understand muscle dystrophy Research Project | 1 Project MembersDuchenne Muscular Dystrophy (DMD) and LAMA2-related Muscular Dystrophy (LAMA2 MD) are devastating rare genetic diseases of childhood manifested by progressive skeletal muscle wasting and ultimately death. In both diseases, the muscle undergoes constant cycles of degeneration-regeneration exacerbated by intrinsic muscle stem cell (MuSC) dysfunction and their impaired ability to support long-term regeneration. Till date, the nature of the cellular changes that contribute to muscle pathologies are unknown. Specifically, there is critical lack of research that provides an unbiased elucidation of the cellular events underlying the different steps of muscle disease progression at the single-cell level. This impedes development of therapeutic strategies that can effectively treat these devastating diseases. Currently, there is no known cure for LAMA2 MD and no therapy that can halt DMD disease progression. The overall goal of our project is to use a combination of single-cell transcriptomics (RNA-sequencing) and proteomics (mass cytometry, CyTOF) to define the cellular composition of diseased muscle tissues at the single-cell resolution. We will delineate the different cell populations that pre-exist and arise during disease progression and classify the novel cellular subsets involved in this process. This data, in combination with genetic lineage tracing will allow reconstruction of the MuSC lineage hierarchy. We will further characterize cellular subpopulations associated with human muscle dystrophies by performing 3-Dimensional intact-tissue RNA sequencing on patient biopsies. Thus, MYOCITY will significantly advance the current understanding of muscle dystrophies, uncover disease-related subsets in human biopsies and also develop new biomarkers and eventually new pharmacological approaches that will stimulate intrinsic muscle tissue repair. Origin and function of signals driving sarcopenia and neuromuscular synapse destabilization Research Project | 1 Project MembersLead Verlust von Muskelmasse und Funktion im Alter führt zu Gangunsicherheiten und dadurch zu einer erhöhten Sturzgefahr. Die molekularen Ursachen dieses «Sarkopenie» genannten Muskelschwunds sind jedoch wenig erforscht. Dieses Projekt möchte einen Beitrag leisten, dessen Ursachen besser zu verstehen. Lay summary Bei einem normal gewichtigen Menschen liegt der Muskelanteil bei bis zu 50% der Körpermasse, kann jedoch durch viele äussere Einflüsse (Sport oder Immobilisierung) verändert werden. Gezieltes Muskelaufbautraining aktiviert einen Signalweg in den Muskelfasern, welcher die Proteinsynthese stimuliert. Ist der gleiche Signalweg jedoch in Muskelfasern von genetisch veränderten Mäusen ständig aktiv, nimmt die Muskelmasse ab und die Tiere entwickeln eine frühzeitige Sarkopenie mit Veränderungen in den gleichen Signalwegen wie bei alten Mäusen. In den geplanten Experimenten werden wir diese Signalwege funktionell untersuchen und die Zellen identifizieren, welche für die altersbedingten Veränderungen verantwortlich sind. Zudem werden wir die Auswirkungen des Alterns auf die Verbindung zwischen Nerven und Muskelfasern genauer untersuchen. Dieses Projekt trägt zu einem besseren Verständnis der Ursachen von Sarkopenie bei. Daraus können in Zukunft vielleicht Therapieansätze entwickelt werden, welche den Verlust an Muskelmasse im Alter verlangsamen oder gar verhindern. Solche Therapien haben das Potential die Lebensqualität der ständig älter werdenden Bevölkerung zu verbessern. The role of mTORC1 in muscle proteostasis Research Project | 2 Project MembersSkeletal muscle accounts for up to 50% of the entire body weight in humans. It is essential for locomotion and breathing and it affects whole-body metabolism. Preservation of muscle mass is thus critical to maintain body function and health. Current views indicate that muscle mass is controlled by the tight balance between protein synthesis and protein degradation, called proteostasis. Perturbation of this balance by extrinsic factors, as for example seen in cachexia (i.e., muscle loss as a secondary consequence of e.g. cancer, AIDS, or cardiac and kidney disease) or in sarcopenia (i.e., loss of muscle mass and function as a consequence of aging), is a main cause of loss of life quality and increased mortality. Thus, a better molecular understanding of muscle proteostasis is of fundamental importance to develop possible treatment strategies to counteract the above diseases. Proteostasis is thought to be controlled (1) by the mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) by regulating protein synthesis (Laplante and Sabatini, 2012) and (2) by forkhead box O (FoxO) transcription factors by regulating expression of the proteins involved in protein degradation (Milan et al., 2015; Zhao et al., 2007). Thus, inhibition of mTORC1 and/or activation of FoxO would cause muscle loss (atrophy), whereas activation of mTORC1 and/or inhibition of FoxO would result in muscle gain (hypertrophy). Consistent with this notion, genetic inactivation of mTORC1 by muscle-specific depletion of the mTORC1-essential component raptor causes muscle atrophy (Bentzinger et al., 2008). However and in striking contrast to the expected outcome of muscle gain, sustained activation of mTORC1 in muscle by knockout of its upstream inhibitor Tsc1 (TSCmKO mice) also results in atrophy, severe myopathy and early death (Bentzinger et al., 2013; Castets et al., 2013). This phenotype is observed despite the marked increase in protein synthesis. Hence, the mechanisms involved in muscle atrophy in TSCmKO mice remains unresolved. Marco Kaiser investigated the mechanisms that may underlie this phenotype during his Ph.D. thesis. In particular, he investigated the role of the most important protein degradation pathway of cells, which is the ubiquitin-proteasome system (UPS). His work has shown that sustained activation of mTORC1 in muscles of TSCmKO mice indeed leads to a significant increase in the expression of all the proteasomal subunits and increased UPS activity. Marco also observed an increase in the activity of the proteasome in lysates of TSCmKO muscles using an in vitro assay. Interestingly, the same increase in proteasome activity was observed in "inducible-TSCmKO" (iTSCmKO) mice upon acute muscle-specific deletion of Tsc1 for 3 weeks. This increase in proteasome activity was, at least in part, reversed by short-term treatment of the TSCmKO mice with the mTORC1-inhibitor rapamycin. Marco also discovered that increased UPS activity was accompanied by a concomitant increase of the transcription factor "nuclear factor, erythroid-derived 2,-like 1" (Nfe2l1, also called Nrf1). Moreover, he found evidence that Nrf1 may regulate the UPS in TSCmKO mice. We now aim to establish that this increase in UPS activity is the mechanism that causes muscle atrophy in TSCmKO mice and to test the functional role of Nrf1 in the regulation of the UPS. To do this, we will perturb the function of the UPS by bortezomib (BTZ) and by knocking down Nrf1 by shRNA in skeletal muscle in vivo. We hope that these experiments will firmly establish that mTORC1 activation is the main driver of protein degradation in skeletal muscle and that Nrf1 is the main regulator of this pathway. These experiments are important to better understand the control of muscle mass and to eventually develop new therapeutic agents that could slow-down the massive muscle wasting observed in cachexia and sarcopenia. 1234 1...4
Deciphering the molecular basis of selective muscle vulnerability in Myasthenia Gravis Research Project | 1 Project MembersImported from Grants Tool 4702817
Characterization of pathophysiological mechanisms involved in the selective vulnerability of muscles in Myasthenia Gravis Research Project | 2 Project MembersNo Description available
Locations, interactions, functions and expression (LIFE) of signals involved in sarcopenia Research Project | 1 Project MembersNo Description available
Sinergia: Machine-learned Design and Bioxolography of Functional 3D Skeletal Muscle Tissues Research Project | 1 Project MembersEngineered 3D skeletal muscle tissue (SMT) is an important tool to study muscle physiology and disease. SMT engineering has applications in regenerative medicine, in vitro drug screening, bio-hybrid robotics, and cultured meat. However, state-of-the-art engineered muscle tissue does not effectively mimic the cellular heterogeneity, architecture, and performance of biological muscle. To address this, researchers are developing techniques to bioprint, differentiate, and mature functional muscle architectures. Machine Learning (ML) approaches could streamline SMT design by rapidly exploring the ideal conditions for muscle biofabrication, computationally capturing the complex interplay between biofabrication parameters (bioprinting, differentiation, and mechanical and electrical maturation), and the resulting functionality of the contractile SMT. This project consists of four key objectives: (1) Adaptation of xolography1 to muscle bioprinting. We will develop the bioxolography technique to fabricate highly aligned and anisotropic cell-laden hydrogels without constraints on the achievable shapes. (2) Biofabrication and differentiation of 3D heterocellular muscle constructs. We will develop a protocol to bioprint, culture, and differentiate arrays of muscle bundles which contain multiple cell types, thereby mimicking the natural muscle. (3) Maturation and characterization of engineered muscle tissues. We will mechanically stimulate our engineered muscle tissues for maturation and characterize their cellular morphology and contractile performance. (4) Development of an ML pipeline to guide the biofabrication and design of muscle actuators. Finally, we will develop an ML pipeline which maps biofabrication and tissue engineering parameters to performance metrics of engineered muscle (structure and function), leveraging a differentiable simulation approach originally developed to model soft material and actuator deformation in soft robotics. We will demonstrate proof-of-concept of this model by designing a centimeter-scale asymmetric, antagonistically actuated skeletal muscle construct. Methods. (1) We will print highly aligned, multinucleated and large (2 to 3-cm-long) bundles of muscle fibers by developing bioxolography: a linear volumetric bioprinting process (LVBP), realized by designing new photoresins and photoinitiators for previously established xolographic printing. (2) Primary murine myoblasts will be co-printed with fibro-adipogenic progenitors (FAPs), and cell culture will be optimized to promote myogenesis, myogenic cell differentiation, and tissue maturation in three dimensional tissue. (3) Muscle tissue will be mechanically stimulated under varying conditions, and characterized for structure and response to electrical stimulation. In a further iteration, we will also characterize muscle tissue co-cultured with optogenetically modified motor neurons to realize a light-controllable innervation system for remote neural actuation of muscle tissue. (4) We will create an in-silico-to-in-vitro platform to optimize muscle design by using ML and our differentiable finite element method (FEM) to map biofabrication parameters to SMT's contractility. This project will accelerate and improve the design and fabrication of functional SMTs by providing an in-silico-to-in-vitro platform for 3D muscle construct design. Our work will provide insights into biofabrication, soft materials, 3D cell culture techniques, hetero-cellular models, bio-hybrid technologies, robotic design, and biophysical cell stimulation. We anticipate multidisciplinary advancements in tissue engineering, muscle physiopathology, 3D bioprinting, the development of new therapeutics, biological machine learning, bio-hybrid robotics, and engineering with living materials.
Role of Fam134b-driven endoplasmic reticulum (ER)-phagy in sarcopenia Research Project | 1 Project MembersAutophagy is a process by which cells remove damaged organelles or protein aggregates to maintain cellular homeostasis. ER-phagy is a recently identified form of autophagy, which clears damaged parts of ER membrane. To elicit ER-phagy, specific ER-bound proteins recognize ER damage and recruit autophagy machinery. The ER is particularly interesting in myofibers because it adopts a unique structure of a very thin sheet attached underneath the plasma membrane. Proper ion flux through muscle ER is essential for contraction. Also, ER is an integral component of anchoring myonuclei at the cell periphery. Despite the obvious importance of the ER in myofibers, it is unknown how the quality of the ER is controlled and whether it is affected during ageing. From snRNA-Seq studies, a novel nuclear population was identified in aged muscles that included Fam134b as a distinguishing marker gene. Our consortium independently confirmed that Fam134b transcripts are increased during muscle ageing in bulk RNA-seq (Börsch et al., 2021; Fig. 1B) and snRNA-FISH (Fig. 1C). We hypothesize that increased Fam134b expression drives ER-phagy during muscle ageing and, importantly, that manipulation of ER-phagy can mitigate sarcopenia. To begin to tackle this question, we have joined forces between two groups with expertise on myonuclear gene expression (Kim) and muscle ageing and mTORC1 signaling (Rüegg), respectively. With the support of Eucor_seed, we aim to obtain foundational data to strengthen our hypothesis and secure larger future funding. Specifically, we will 1) test our core idea that ER-phagy flux is altered in aged muscles and examine its relationship to the mTORC1 pathway, 2) investigate how modulating Fam134b expression affects muscle performance in young and old mice, and 3) take the first step in identifying upstream regulators of Fam134b expression during ageing.
Spatial transcriptomics: Novel methods to detect pre-symptomatic causes of Sarcopenia Research Project | 2 Project MembersAs overall life expectancy increases so do comorbidities associated with an ageing population. Sarcopenia, the progressive loss of skeletal muscle mass and function, is widespread amongst the elderly and leads to reduced quality of life, frailty, and ultimately death - representing a major public health issue and clinical challenge. Sarcopenia entails complex pathogenesis involving neuromuscular function, and metabolism changes are suggested to be the first sign of muscle ageing. Thus, understanding these could help detect pre-symptomatic sarcopenia. The multinucleated composition of myofibers has hampered single-cell analysis, but efforts in bulk, single-nuclear RNA-seq and multiplex-FISH identified sarcopenic changes originating from differences in cell populations [1]. Therefore, the only methodology able to pinpoint the sub-cellular and cell-specific localisation of signals driving sarcopenia is spatial transcriptomics [2-3]. Most workflows however have limited gene coverage, and some proprietary methods with higher capability seem not to be fully reproducible. Therefore, the research community requires openly accessible protocols allowing the resolution of >10,000 genes at sub-diffraction-limit which facilitates distinction of transcripts in immediate proximity, to further encourage innovation in sarcopenic research and ultimately healthcare. This project aims to address this and will create methods, including detailed instructions on probe design, available for others to reproduce.
Linker-based gene therapy of LAMA2-related muscular dystrophy using AAV-MYO Research Project | 1 Project MembersExtracellular matrix (ECM) acts as scaffold to provide essential structural support to the surrounding cells. Laminins are essential components of the ECM connecting it tightly with the underlying cell layer, just like the binds between scaffolding and a building. Mutations in this protein family cause a range of severe disorders that affect different organs. The rope-like laminin-211 protein, composed of three intertwined laminin chains, is the main isoform in skeletal muscle. Mutations in the LAMA2 gene, encoding one of the chains in laminin-211, leads to its loss and causes a rare, severe and early-onset muscular dystrophy, called MDC1A or LAMA2 MD. Affected children show muscle wasting and weakness and eventually die from respiratory insufficiency. There is no treatment for LAMA2 MD. We have shown that two specifically designed linker proteins strongly improve the pathology and lifespan in LAMA2 MD mice, which lack laminin-211 like LAMA2 MD patients and display very similar symptoms. In ongoing research, we are developing a gene therapy approach to deliver the two linkers to LAMA2 MD mice by using an adeno-associated viral vector (AAV9). Our initial results show that this gene therapy approach is indeed possible. We now aim to optimize linker delivery using newly developed AAV9 variants (myotropic AAVs) that are even more efficient at infecting skeletal muscles. We are hopeful these experiments will pave the way to a gene therapy treatment for LAMA2 MD patients.
A multidimensional single-cell approach to understand muscle dystrophy Research Project | 1 Project MembersDuchenne Muscular Dystrophy (DMD) and LAMA2-related Muscular Dystrophy (LAMA2 MD) are devastating rare genetic diseases of childhood manifested by progressive skeletal muscle wasting and ultimately death. In both diseases, the muscle undergoes constant cycles of degeneration-regeneration exacerbated by intrinsic muscle stem cell (MuSC) dysfunction and their impaired ability to support long-term regeneration. Till date, the nature of the cellular changes that contribute to muscle pathologies are unknown. Specifically, there is critical lack of research that provides an unbiased elucidation of the cellular events underlying the different steps of muscle disease progression at the single-cell level. This impedes development of therapeutic strategies that can effectively treat these devastating diseases. Currently, there is no known cure for LAMA2 MD and no therapy that can halt DMD disease progression. The overall goal of our project is to use a combination of single-cell transcriptomics (RNA-sequencing) and proteomics (mass cytometry, CyTOF) to define the cellular composition of diseased muscle tissues at the single-cell resolution. We will delineate the different cell populations that pre-exist and arise during disease progression and classify the novel cellular subsets involved in this process. This data, in combination with genetic lineage tracing will allow reconstruction of the MuSC lineage hierarchy. We will further characterize cellular subpopulations associated with human muscle dystrophies by performing 3-Dimensional intact-tissue RNA sequencing on patient biopsies. Thus, MYOCITY will significantly advance the current understanding of muscle dystrophies, uncover disease-related subsets in human biopsies and also develop new biomarkers and eventually new pharmacological approaches that will stimulate intrinsic muscle tissue repair.
Origin and function of signals driving sarcopenia and neuromuscular synapse destabilization Research Project | 1 Project MembersLead Verlust von Muskelmasse und Funktion im Alter führt zu Gangunsicherheiten und dadurch zu einer erhöhten Sturzgefahr. Die molekularen Ursachen dieses «Sarkopenie» genannten Muskelschwunds sind jedoch wenig erforscht. Dieses Projekt möchte einen Beitrag leisten, dessen Ursachen besser zu verstehen. Lay summary Bei einem normal gewichtigen Menschen liegt der Muskelanteil bei bis zu 50% der Körpermasse, kann jedoch durch viele äussere Einflüsse (Sport oder Immobilisierung) verändert werden. Gezieltes Muskelaufbautraining aktiviert einen Signalweg in den Muskelfasern, welcher die Proteinsynthese stimuliert. Ist der gleiche Signalweg jedoch in Muskelfasern von genetisch veränderten Mäusen ständig aktiv, nimmt die Muskelmasse ab und die Tiere entwickeln eine frühzeitige Sarkopenie mit Veränderungen in den gleichen Signalwegen wie bei alten Mäusen. In den geplanten Experimenten werden wir diese Signalwege funktionell untersuchen und die Zellen identifizieren, welche für die altersbedingten Veränderungen verantwortlich sind. Zudem werden wir die Auswirkungen des Alterns auf die Verbindung zwischen Nerven und Muskelfasern genauer untersuchen. Dieses Projekt trägt zu einem besseren Verständnis der Ursachen von Sarkopenie bei. Daraus können in Zukunft vielleicht Therapieansätze entwickelt werden, welche den Verlust an Muskelmasse im Alter verlangsamen oder gar verhindern. Solche Therapien haben das Potential die Lebensqualität der ständig älter werdenden Bevölkerung zu verbessern.
The role of mTORC1 in muscle proteostasis Research Project | 2 Project MembersSkeletal muscle accounts for up to 50% of the entire body weight in humans. It is essential for locomotion and breathing and it affects whole-body metabolism. Preservation of muscle mass is thus critical to maintain body function and health. Current views indicate that muscle mass is controlled by the tight balance between protein synthesis and protein degradation, called proteostasis. Perturbation of this balance by extrinsic factors, as for example seen in cachexia (i.e., muscle loss as a secondary consequence of e.g. cancer, AIDS, or cardiac and kidney disease) or in sarcopenia (i.e., loss of muscle mass and function as a consequence of aging), is a main cause of loss of life quality and increased mortality. Thus, a better molecular understanding of muscle proteostasis is of fundamental importance to develop possible treatment strategies to counteract the above diseases. Proteostasis is thought to be controlled (1) by the mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) by regulating protein synthesis (Laplante and Sabatini, 2012) and (2) by forkhead box O (FoxO) transcription factors by regulating expression of the proteins involved in protein degradation (Milan et al., 2015; Zhao et al., 2007). Thus, inhibition of mTORC1 and/or activation of FoxO would cause muscle loss (atrophy), whereas activation of mTORC1 and/or inhibition of FoxO would result in muscle gain (hypertrophy). Consistent with this notion, genetic inactivation of mTORC1 by muscle-specific depletion of the mTORC1-essential component raptor causes muscle atrophy (Bentzinger et al., 2008). However and in striking contrast to the expected outcome of muscle gain, sustained activation of mTORC1 in muscle by knockout of its upstream inhibitor Tsc1 (TSCmKO mice) also results in atrophy, severe myopathy and early death (Bentzinger et al., 2013; Castets et al., 2013). This phenotype is observed despite the marked increase in protein synthesis. Hence, the mechanisms involved in muscle atrophy in TSCmKO mice remains unresolved. Marco Kaiser investigated the mechanisms that may underlie this phenotype during his Ph.D. thesis. In particular, he investigated the role of the most important protein degradation pathway of cells, which is the ubiquitin-proteasome system (UPS). His work has shown that sustained activation of mTORC1 in muscles of TSCmKO mice indeed leads to a significant increase in the expression of all the proteasomal subunits and increased UPS activity. Marco also observed an increase in the activity of the proteasome in lysates of TSCmKO muscles using an in vitro assay. Interestingly, the same increase in proteasome activity was observed in "inducible-TSCmKO" (iTSCmKO) mice upon acute muscle-specific deletion of Tsc1 for 3 weeks. This increase in proteasome activity was, at least in part, reversed by short-term treatment of the TSCmKO mice with the mTORC1-inhibitor rapamycin. Marco also discovered that increased UPS activity was accompanied by a concomitant increase of the transcription factor "nuclear factor, erythroid-derived 2,-like 1" (Nfe2l1, also called Nrf1). Moreover, he found evidence that Nrf1 may regulate the UPS in TSCmKO mice. We now aim to establish that this increase in UPS activity is the mechanism that causes muscle atrophy in TSCmKO mice and to test the functional role of Nrf1 in the regulation of the UPS. To do this, we will perturb the function of the UPS by bortezomib (BTZ) and by knocking down Nrf1 by shRNA in skeletal muscle in vivo. We hope that these experiments will firmly establish that mTORC1 activation is the main driver of protein degradation in skeletal muscle and that Nrf1 is the main regulator of this pathway. These experiments are important to better understand the control of muscle mass and to eventually develop new therapeutic agents that could slow-down the massive muscle wasting observed in cachexia and sarcopenia.
