Periodic Reporting for period 2 - Myo_LysoZOOM (An insight into lysosomal signature in muscle wasting)
Reporting period: 2023-07-01 to 2024-06-30
Muscle wasting is the progressive loss of skeletal muscle mass caused by immobility, ageing, malnutrition, medications, injuries, or diseases that impact the musculoskeletal or nervous system. Severe muscle loss translates into muscle weakness and frailty, leading to poor quality of life along with increased disability, morbidity and mortality. As a result, muscle wasting is a significant public health burden deeply affecting welfare and healthcare costs.
Recent evidence showed that attenuating muscle wasting in disease conditions ameliorates prognosis and survival; nevertheless, to date a proper strategy to fully and transversally prevent muscle wasting is missing, reflecting the fragmented knowledge of the signals at the basis of the phenomenon. For these reasons, a deeper comprehension of the molecular mechanisms involved will be fundamental for the discovery and characterization of novel potential targets for drug development.
Muscle wasting is indeed a complex phenomenon that involves the activation of several interconnected signaling events and pathways, although not all of them have been sufficiently characterized. With the intention of filling this gap, the project aims to expand the knowledge of the molecular mechanisms controlling muscle homeostasis and to unravel the connection between lysosomal signaling and muscle atrophy.
Therefore, Myo_LysoZOOM main objective has been to investigate the relevance of unknown lysosome-based signals for muscle physiology in healthy and wasting conditions. This has been possible thanks to the development of a new methodology allowing the rapid isolation of lysosomes from fresh tissues that were subsequently profiled for polar metabolites and proteins content in different muscle wasting conditions and at different timepoints. The generated datasets have been at the basis for the identification of the common lysosomal signature impacting muscle mass homeostasis. Interestingly, lysosomal glycolysis has been shown to be fundamental to guarantee key lysosomal processes that are the basis of lysosal homeostasis and function.
In conclusion, the project strongly contributed to the generation of novel knowledge on a complex phenomenon deeply affecting modern society, with the potential of being the basis for new therapies development.
Moreover, through the accomplishment of the action and the establishment of this new research line, Myo_LysoZOOM also fostered Dr. Armani’s scientific and professional growth, that guided the researcher towards a more independent position with the acquiring of independent funding for continuing his own research .
Recent evidence showed that attenuating muscle wasting in disease conditions ameliorates prognosis and survival; nevertheless, to date a proper strategy to fully and transversally prevent muscle wasting is missing, reflecting the fragmented knowledge of the signals at the basis of the phenomenon. For these reasons, a deeper comprehension of the molecular mechanisms involved will be fundamental for the discovery and characterization of novel potential targets for drug development.
Muscle wasting is indeed a complex phenomenon that involves the activation of several interconnected signaling events and pathways, although not all of them have been sufficiently characterized. With the intention of filling this gap, the project aims to expand the knowledge of the molecular mechanisms controlling muscle homeostasis and to unravel the connection between lysosomal signaling and muscle atrophy.
Therefore, Myo_LysoZOOM main objective has been to investigate the relevance of unknown lysosome-based signals for muscle physiology in healthy and wasting conditions. This has been possible thanks to the development of a new methodology allowing the rapid isolation of lysosomes from fresh tissues that were subsequently profiled for polar metabolites and proteins content in different muscle wasting conditions and at different timepoints. The generated datasets have been at the basis for the identification of the common lysosomal signature impacting muscle mass homeostasis. Interestingly, lysosomal glycolysis has been shown to be fundamental to guarantee key lysosomal processes that are the basis of lysosal homeostasis and function.
In conclusion, the project strongly contributed to the generation of novel knowledge on a complex phenomenon deeply affecting modern society, with the potential of being the basis for new therapies development.
Moreover, through the accomplishment of the action and the establishment of this new research line, Myo_LysoZOOM also fostered Dr. Armani’s scientific and professional growth, that guided the researcher towards a more independent position with the acquiring of independent funding for continuing his own research .
The entire project has been based on the development of a novel methodological approach to isolate lysosomes from fresh murine muscles; to achieve this, a murine genetic model has been developed, allowing the transgenic expression of a tagged protein on the surface of lysosomes only in skeletal muscle. This model enabled the establishment of a protocol suitable for the immunopurification of organelles starting from muscle tissue of different atrophic models. In detail, optimization has been performed in order to collect organelles that were not broken during the isolation procedure, that were pure from other cellular compartments and that were able to retain their content and function. All these steps were successfully achieved and allowed the progression of the project towards datasets generation. After method validation, organelles were isolated from atrophic muscles from different models at two timepoints; this allowed to capture different signaling events happening in atrophic conditions with diverse etiologies in order to identify the common lysosomal signature contributing to muscle wasting. To do that, lysosomes were profiled for their content in terms of polar metabolites and proteins. In particular, this kind of analysis unraveled the dynamics of these organelles in response to wasting stimuli by highlighting internal molecules or surface interactors variations in relation to deviations from normal physiology. These datasets have been produced and a list of metabolites and proteins has been generated per each condition analyzed.
Three major cellular pathways have been identified as lysosomal signatures in muscle wasting. However, only glycolysis on the lysosomal fraction has been mechanically investigated. Thanks to the generation of a GAA knockout/LysoTag model in which glucose homeostasis is perturbed by the lack of lysosomal glycogen degradation, the project was able to identify and mechanistically characterized the functional meaning of lysosomal surface glycolytic microdomains. to sustain fundamental lysosomal activities like vesicle fusion/organelles tethering and more importantly V-ATPase activity and the consequent lysosomal function.
As a final remark, this project and its results have and will have a series of different high impact implications: 1. they expanded the comprehension of the biology of the complicated wasting phenomenon that can be exploited by the muscle community; 2. they fostered and propelled the independence of a young researcher towards the establishment of a revolutionary and innovative research line that will define his career path; 3. the results are providing and will provide a powerful resource and platform for the identification of new potential pharmacological targets that can be exploited by a broader scientific community.
Three major cellular pathways have been identified as lysosomal signatures in muscle wasting. However, only glycolysis on the lysosomal fraction has been mechanically investigated. Thanks to the generation of a GAA knockout/LysoTag model in which glucose homeostasis is perturbed by the lack of lysosomal glycogen degradation, the project was able to identify and mechanistically characterized the functional meaning of lysosomal surface glycolytic microdomains. to sustain fundamental lysosomal activities like vesicle fusion/organelles tethering and more importantly V-ATPase activity and the consequent lysosomal function.
As a final remark, this project and its results have and will have a series of different high impact implications: 1. they expanded the comprehension of the biology of the complicated wasting phenomenon that can be exploited by the muscle community; 2. they fostered and propelled the independence of a young researcher towards the establishment of a revolutionary and innovative research line that will define his career path; 3. the results are providing and will provide a powerful resource and platform for the identification of new potential pharmacological targets that can be exploited by a broader scientific community.
Myo_LysoZOOM is an ambitious project that aimed to cover major gaps in the comprehension of the molecular mechanisms at the basis of muscle wasting. In particular, the relevance of lysosomes in the atrophy process was never addressed before due to technical challenges in the isolation of these organelles. For these reasons, the development of the genetic model and the optimization of a novel method to isolate lysosomes are the first major achievements overcoming the limitations in the study of lysosomal contribution to muscle atrophy. Moreover, all the datasets produced until now are the first available data on lysosomal dynamics during atrophy, representing a powerful resource towards the comprehension of lysosomal signaling in muscle wasting.
Final goal of the project has been the identification of novel signaling events stemming from lysosomes and contributing to muscle loss in different conditions. Surprisingly, all 10 glycolytic enzymes are specifically enriched in muscular lysosomes not only in wasting conditions but already in healthy skeletal muscles at rest. This is so far the most exciting and revolutionary discovery from this study, demonstrating the presence of a fundamental metabolic pathway (glycolysis) in close physical proximity to lysosomes, thus demonstrating the potential presence of glycolysis function microdomains on the surface of lysosomes.
Overall, the results of this project will have a series of different high impact implications: 1. they expanded the comprehension of the biology of the complicated muscle wasting phenomenon with potential for future drug developments; 2. they fostered the understanding of lysosomes biology in the physiology of a tissue; 3. they provided a powerful resource for the muscle community in order to investigate the biology of this tissue from a completely novel perspective.
As a whole, the proposed Myo_LysoZOOM project will produce a long-term extraordinary impact on European research and innovation, science and society.
Final goal of the project has been the identification of novel signaling events stemming from lysosomes and contributing to muscle loss in different conditions. Surprisingly, all 10 glycolytic enzymes are specifically enriched in muscular lysosomes not only in wasting conditions but already in healthy skeletal muscles at rest. This is so far the most exciting and revolutionary discovery from this study, demonstrating the presence of a fundamental metabolic pathway (glycolysis) in close physical proximity to lysosomes, thus demonstrating the potential presence of glycolysis function microdomains on the surface of lysosomes.
Overall, the results of this project will have a series of different high impact implications: 1. they expanded the comprehension of the biology of the complicated muscle wasting phenomenon with potential for future drug developments; 2. they fostered the understanding of lysosomes biology in the physiology of a tissue; 3. they provided a powerful resource for the muscle community in order to investigate the biology of this tissue from a completely novel perspective.
As a whole, the proposed Myo_LysoZOOM project will produce a long-term extraordinary impact on European research and innovation, science and society.