Dr TSAI Shih-Yin

Principal Investigator

Email: phsts@nus.edu.sg

Qualification: Ph.D. (Molecular Genetics. The Ohio State University, USA)

Linkedin: www.linkedin.com/in/shihyin-tsai-266b5959

ORCID: orcid.org/0000-0002-2834-6383

HUANG Chien-Yung

Research Associate

Email: phshc@nus.edu.sg

Qualification: M.Sc. (Biochemistry and Molecular Biology. National Yang Ming University, Taiwan)

Project Description: There are many aging related diseases that leads to mortality. One of major causes is sarcopenia, which comes with gradual loss of skeletal muscle mass. With the progression of research, aging is no longer considered as an inevitable scenario. One of the molecular pathways leading the aging is the mTOR pathway. The elderly display higher mTORC1 and its downstream S6K1 activities in the skeletal muscle. The mice with mTOR activation in muscle (TSC1mKO) exhibited late on-set of myopathies but reduced lifespan compared to the control. While the reduction of mTOR downstream activity (S6K1mKO) displayed the opposite phenotypes. My main project is to dissect the molecular mechanism of mTOR activities affecting the downstream phenotypes in the cellular level. I would also like to understand how S6K1 regulates its downstream effectors during metabolic stress such as exercise.

Yifan LYU

Visiting Scientist

Email: phsv67@visitor.nus.edu.sg

Qualification: MD, PhD (Gerontology, Nanjing Medical University, China)

Project description: Project description: Sarcopenia is a condition which is defined by the gradual loss of skeletal muscle mass and function with age. Normally, Skeletal muscle homeostasis is strongly dependent on protein degradation. The two major intracellular protein degradation systems are the ubiquitin-proteasome system and autophagy-lysosome system. In my project, I establish in vitro models to demonstrate the role of autophagy-lysosome system and Ubiquitin-proteasome system during myoblast differentiation. The myocyte with damaged organelle aggregation would not become mature myotube. Additionally, mTORC1, a mTOR complex which has been shown to play an important role in protein degradation. I also observe the alternation of mTORC1 signal pathway in myoblast differentiation. Thus, the goal of the project is to investigate the molecular mechanism that mediating protein degradation during myoblast differentiation and try to find a target that can maintain muscle homeostasis.

KOH Shu Wen

Laboratory Technologist

Email: phsksw@nus.edu.sg

Qualification: B.Sc. (Biomedical Science, UWA)

Description: Shu Wen’s main responsibility is to ensure smooth running of the laboratory. This includes procurement of laboratory’s supplies, tracking of laboratory finances, safety related issues, administration and day to day laboratory operation.

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LAUWERS Marianne Marie E

Research Fellow

(July 2023 - July 2024)

Qualification: PhD (Veterinary sciences, UGent)

Project description: The proportion of elderly in the world’s population is ever-growing and estimated to constitute about 25% by 2050. This will increase the burden of age-related diseases on the health care system and grow the need for new treatment approaches. Physical dysfunction as result of musculoskeletal diseases such as sarcopenia is of particular interest as it will lead to disability, immobility and loss of independence, increasing the burden of other chronic diseases.
Previously, our group showed that the muscle specific activation of 4EBP1 leads to an increase of post-synaptic myonuclei and a surge in the number and activity of satellite cells in ageing mice as well as mice with a sarcopenic phenotype. Thereby rescuing two distinct characteristics of muscle ageing.
In my project, the goal is to develop a 3D in vitro model including key physiological and pathological features and evaluate the molecular mechanisms behind sarcopenic and ageing muscles. More specifically, the aim is to further elaborate the role of 4EBP1 in the recruitment of post-synaptic myonuclei and satellite cells as well as investigate if muscle specific 4EBP1 activation can also rescue muscle ageing induced by adipose senescence or fibrosis.
Finally, the possibility to mimic the effect of muscle specific 4EBP1 activation will be investigated in ageing mice by inducing a healthy ageing physiology, such as calorie restriction or exercise.

DONG Han

Graduate Student

(August 2019 - May 2024)

Project Description: Loss of lean mass is a common phenomenon in aged people, named sarcopenia. Sarcopenia is highly associated with the development of insulin resistance and causing Type II Diabetes. The primary risk factor of sarcopenia is age. However, obesity, the accumulation of adipose tissue, increases the risk of developing sarcopenia by, termed “obesity sarcopenia”. By doing exercise, people preserve their muscle mass and reduce the accumulation of fat mass at the same time. So, my project is focusing on the contact between skeletal muscle and adipose tissue. Previous studies have shown that genetically modified skeletal muscle often affects the changes in the whole-body lipid metabolism, which in turn affects the homeostasis of adipose tissue, which indicated that the muscle-adipose contact does exist. I am interested in the molecular mechanism of muscle-adipose communication and how this contact function in the aging process.

Dhivya SANKARANARAYANAN

Research Assistant

(November 2022 - November 2023)

Project Description: The 4EBP1 regulates the protein quality control in skeletal muscle. My project involves measuring the mRNA expression level of genes related with protein degradation pathways in wild type and 4EBP1 mutant-muscle mice gastrocnemius muscle under fed and 48 hours starvation conditions. Also, I am separating and testing the 2 protein degradation pathways such as ubiquitin proteasome system and autophagy lysosome system and analyzing how these are altered by 4EBP1 and by which stage.

Bindu Priyanka BAVANI RAJAN

Research Assistant

(October 2022 - October 2023)

Project Description: Morphology changes in tissues are often indications of the disease process. I am exploring the techniques and analysis in histology and pathology to study aging, particularly in skeletal muscle. My project involves developing various specialized staining, to analyse muscle metabolic capacities, such as Periodic Acid-Schiff Stain for glycogen deposit and Succinic Dehydrogenase Histochemical staining for analysis of mitochondrial activities and to characterise muscle fibre types such as immunofluorescence analysis for myosin heavy chain expression.

Elisa Marie CROMBIE

Graduate Student

(August 2017 - August 2022)

Project Description: Mammalian target of rapamycin complex 1 (mTORC1) is central in a pathway integrating nutrient sensing from the environment for the promotion of anabolic signals in the cell including protein synthesis, cell growth and survival, as well as inhibiting autophagy. Dysregulation of mTORC1 signalling is seen with ageing; therefore, my project is focussing on developing an ageing mouse model of increased mTORC1 signalling in muscle (TSC1mKO) and understanding its associated pathology. Previously, TSC1mKO mice have been shown to exhibit leanness and resistance to obesity at a young age, but show myopathy at older age, with a shorter lifespan than wild-type controls. I am interested to identify the molecular mechanisms contributing to this phenotype and corresponding effects on whole-body metabolism and autophagic regulation.

Anna Elizabeth KOZLOV

Amgen Scholars Internship Student, Emory University

(June 2022 to August 2022)

Project Title: Mass Up Aging Skeletal Muscle study of mTORC1 and sarcopenia

Project Description: Sacropenia is an age-associated disorder marked by gradual loss of skeletal muscle mass. mTORC1, a mTOR complex which has been shown to play an important role in protein homeostasis, has emerged as an important pathway for the maintenance of skeletal muscle mass with age. Mice with chronic mTOR activation (TSC1mKO) exhibit late on-set pathologies which are largely reversed by the downstream activation of 4EBP1. 4EBP1 not only decreased protein synthesis, but also affected autophagic flux suggesting additional modes of mTORC1 regulation of lysosomes besides ULK1 and TFEB/TFE3 pathways. I hypothesize that these additional mechanisms are through translational regulation of lysosome-associated proteins and regulation of lysosome acidification through the localization of mTORC1 complex of lysosome. Thus, my project is focused on further exploring how mTORC1 mediates regulation of protein synthesis in vitro primarily through the use of immunoblotting.

FUNG Wai Ho, Kyle

Internship Student, University of Toronto - St. George Campus

(May 2022 to July 2022)

Project Title: Investigating the role of 4EBP1 in the regulation of autophagy

Project Description: Sarcopenia is the loss of skeletal muscle mass, integrity and function commonly related to aging and is a major risk factor for mortality. Deficient autophagy has been shown to lead to skeletal muscle dysfunction and disorganisation. Chronic activation of mammalian target of rapamycin complex 1 (mTORC1), a regulator of protein homeostasis, has been documented to be associated with aging-related sarcopenia. Upon mTORC1 suppresses autophagy, induces translation initiation via activation of S6 kinase and inhibition of 4EBP1, and promotes skeletal muscle growth. In this lab, 4EBP1 overexpression was previously found to rescues autophagy deficiency caused by mTORC1 overexpression. My project involves processing and immunostaining muscles of transgenic mice models to investigate the mechanisms by which 4EBP1 regulates autophagy or other protein degradation pathways.

LIEW Li Hui

UROPS Student, NUS Life Sciences

(August 2021 - April 2022)

Project title: Investigating the role of S6K1 and TSC1 in the regulation of cardiac hypertrophy with age

Project description: Cardiovascular diseases are the leading cause of death worldwide. A prominent feature of diseased patients is left ventricular hypertrophy, which coincidentally is also a hallmark of an aging heart. Research has also shown that there is chronic activation of mTORC1 signalling in aged mice. I am interested in understanding the roles of S6K1 and TSC1 in the regulation of cardiac hypertrophy in aged organisms. As such, in my project, I use mice models to observe how the deletion of TSC1 and S6K1, which is upstream and downstream of mTORC1 signalling pathway respectively, affects the heart. I do so through using a series of histological methods to process mice tissue; immunohistochemistry methods for staining and analysis of mice tissue; and echocardiography data to understand the effects of the respective protein knockdown on the mice's cardiac functions.

KIM Seonyoung

Research Associate

(June 2020 - February 2022)

Project Description: The mammalian target of rapamycin (mTOR) is well known as crucial regulator for various cellular functions including cell cycle, apoptosis, glucose metabolism, protein synthesis and autophagy. Recently, a growing evidence suggests that mTOR signalling influences longevity and aging. Among targets of mTOR, ribosomal protein S6 kinase 1 (S6K1) is a major downstream signalling molecule of mTOR complex1 and its knockout mice have increased lifespan and improvement in age-related phenotypes, and resistance to type 2 diabetes. Here we identify the effects of S6K1 deletion in skeletal muscle by observing muscle fiber type distribution and atrophy and evaluating exercise performance. Furthermore, we elucidate the characterization of S6K1 in aging model.

ANG Seok Ting Jamie

Research Fellow

(January 2019 – November 2021)

Project Description: Sex differences are apparent in lifespan and aging-associated diseases in humans. For example, depression, a debilitating mood disorder amongst the elderly, is twice more likely to be diagnosed in females than males. Depression is characterised by lack of motivation, extreme fatigue and psychomotor retardation, and is a rising public health burden. The World Health Organization predicts that by 2020, depression will be the second-leading cause of disability in the world, just behind cardiovascular disease, especially with a rapidly rising aging global population. Physical exercise has been proven to be an all-natural and effective treatment to alleviate depression symptoms. The positive effects of exercise suggest that the identification of pathways of communication between the skeletal muscle and brain could lead to discovery of novel treatments to fight depression. Thus, my project will investigate how muscle and neuropsychiatric functions interact during the process of aging. I hypothesise that (1) there is a co-morbid relationship between aging-associated muscle wasting (i.e. sarcopenia) and depression, (2) neuropsychiatric health is dependent on cohesive interactions between the skeletal muscle and brain, and (3) sarcopenia contributes to depression in males and females differently.

Rhea KHANNA

FYP Student, NUS Life Sciences

(August 2020 - April 2021)

Project Title: Investigating the relationship between mTORC1-4EBP1 signalling and NMJ structural stability

Project Description: The mTORC1 signaling pathway regulates many cellular processes such as protein translation via its two downstream targets S6K1 and 4EBP1. More notably, previous studies have shown that mTORC1 signalling regulates structural stability of the neuromuscular junction (NMJ), which is a chemical synapse between a motor neuron and skeletal muscle required for muscle contraction. Preliminary data suggests that it is mTORC1 to 4EBP1 signaling specifically that is responsible for regulating NMJ stability, not S6K1. Transgenic expression of 4EBP1mt-mus mice, where mTORC1/4EBP1 signalling is inhibited, generated numerous tiny fragments of acetylcholine receptor (AChR) clusters even in young mice, resembling that of normal aged NMJs. Furthermore, there was an increased number of postsynaptic myonuclei in 4EBP1mt-mus mice than in the control mice. These observations signify that mTORC1 signalling to 4EBP1 does indeed regulate NMJ stability and reorganisation. However, the exact mechanisms behind these phenomena are yet to be elucidated. Thus, my project will investigate the mechanisms behind how the mTORC1/4EBP1 pathway regulates NMJ stability and morphology, using techniques such as RNA in situ hybridisation with the RNAscope assay to track AChR turnover.

LIM Moon Sim, Gwen

UROPS & FYP Student, NUS Life Sciences

(December 2018 - November 2020)

Project Title: Mapping S6K1 Functionality Loss to Effects on Insulin Sensitivity in Muscles

Project Description: As one of the downstream effectors, activation of S6 kinase 1(S6K1) via PI3-kinase/Akt/mTOR pathway promotes protein synthesis and cellular growth. Yet, in recent years, S6K1 functionality has been expanded to a critical player in the regulation of insulin sensitivity. With the use of S6K1 knockout and different dietary conditions, my project aims to identify effects of S6K1 functionality loss on upstream targets of the PI3-kinase/Akt/mTOR pathway in various muscles under these different environmental factors. This may provide insights to the difference in risks for development of age-related non-communicable diseases such as diabetic myopathy and cardiovascular diseases observed.

Arun S/O GANASARAJAH

Research Assistant

(August 2019 – August 2020)

Project Description: The ribosomal protein S6 kinase beta-1 (S6K1), is part of the mTORC1 signalling pathway that regulates cell growth, motility, and survival. Activation of the mTORC1 signalling pathway results in the phosphorylation of downstream S6K1 which in turn affects protein synthesis. It was demonstrated in 2009 that rapamycin (an inhibitor of mTOR complex) reverses elevated mTORC1 signalling in multiple tissues and rescues skeletal muscle dystrophy. This, as a result, increased the life span of mice and highlighted the role of the mTOR-S6K1 pathway in mammalian aging (Harrison et al., 2009).
Furthermore, in 2017, C.Y. Liao et al. proved that mice survival could be extended by suppressed levels of S6K1. This life extension was observed together with improved muscle function, but not heart or adipose tissue. This was in line with the observation that deletion of S6K1 in muscle tissue specifically extended survival of the mice. Therefore, the rapamycin-induced lifespan extension in mice is in part due to the improvement of skeletal muscle function and can this phenotype can be replicated by reduced S6K1 activity (Liao CY, Anderson SS, Chicoine NH, et al., 2017).
Hence, this study focuses on the muscle-specific deletion of S6K1. S6K1 muscle knock out (S6K1mKO) mice are put through different routines of chronic or acute exercise to elucidate the effects and variation on endurance, strength and physicality throughout the lifespan of the mice.

Pooja SIVA

FYP & Internship student, Republic Polytechnic

(March 2019 - January 2020)

Project Title:The study on the effects of S6K1 and excercising

Project Description: Sarcopenia is highly associated with frailty, which is the major risk of mortality among elderly. Recovery of skeletal muscle function might directly or indirectly improve systemic metabolic decline with aging. Therefore, development of interventions targeting muscle strength and function as treatment for sarcopenia are highly desired. Reduced activity of mTORC1 substrate, ribosomal protein S6 kinase 1 (S6K1), leads to lifespan extension in disparate eukaryotic organisms including mice. Moreover, an increased basal level of phosphorylated mTORC1 and S6K1, has been reported in the elderly human population and may contribute to age-related insulin resistance in skeletal muscle. These findings warrant a more detailed analysis of S6K1 functions pertinent to aging. This project will study how muscle S6K1 regulates metabolic adaptation following metabolic stress and exercise.

GAMUEDA Paul Jerome

URAPS Student, University of Toronto

(May 2019 to August 2019)

Project Title: S6K1 and its Effects on Muscle Fiber Type Distribution and Size

Project Description: Sarcopenia is a process that occurs during aging, leading to loss of muscle mass and function. S6K1 is a factor downstream of the mTOR pathway that is responsible for regulation of muscle size. The purpose of the study was to determine how muscle fiber type distribution and size are affected by knockout of S6K1 in skeletal muscle and by aging.

HERNANDO Adriel

Amgen Scholars Program, Bandung Institute of Technology

(June 2019 to August 2019)

Project Title: Lost In Translation: The Communication between Muscles and Nerves

Project Description: Sarcopenia is a geriatric syndrome that is characterized by the decline in muscle mass and strength. One of the proposed mechanisms underlying the development of sarcopenia is neuromuscular junction (NMJ) degeneration, particularly NMJ denervation. During ageing, muscle weakness and loss of muscle innervation are associated with high activity of the mTORC1 pathway, including increased activity of S6K1, a downstream target of mTORC1 in the muscle. The mTORC1-S6K1 pathway is typically activated during protein synthesis, but its high activity at old age was suggested to mediate ageing-related muscle wasting since inactivation of the pathway in old animals improved symptoms of sarcopenia. Here, using immunohistochemistry and confocal microscopy, I investigated the effect of S6K1mKO on the morphology of aged NMJ.

TAN Kuan Ting

FYP Student, NUS Life Sciences

(August 2018 - April 2019)

Project Title: Relationship between age-associated changes in neuromuscular junction and the mTORC1 pathway

Project Description: The neuromuscular junction (NMJ) refers specifically to the synaptic connection between an axon and a muscle fibre. Apart from electrical signaling from the central nervous system to skeletal muscles, retrograde signaling from the skeletal muscle to the nerves is another crucial mechanism that regulates skeletal muscle growth and sustenance. Previous studies have shown that age-related changes in NMJ may result in increase in muscle atrophy and reduction in retrograde signaling, and while the mTORC1 pathway seems to be involved in these physiological impacts, the mechanisms remain to be elucidated. Hence, through the use of immunofluorescence staining, my project looks at how genetic modifications (4EBP-mt muscle and S6K1 muscle KO) of the mTORC1 pathway may possibly rescue aged NMJ phenotype using sarcopenia TSC1 muscle KO mice model and aged mice. By understanding the relationship between aged NMJ phenotype and corresponding retrograde signaling impact, it provides insights to prevent and reduce age-associated loss in muscle mass and function during sarcopenia at old age.

KHO Yun Suen

UROPS Student, NUS Life Sciences

(July 2017 – April 2018)

Project Title: Effect of S6K1mKO on the Regulation of Whole-body Metabolism

Project Description: S6K1 is a serine kinase downstream from Akt in the insulin signalling pathway, which is essential for the negative feedback regulation of insulin. Research on S6K1 whole body knock out has been shown in various studies to have beneficial effects, such as protection against diet-induced insulin resistance, diet-induced and age-induced obesity and healthier muscles. However, various negative effects on the mice such as beta cells atrophy and impaired short-term memory have been observed. As such, we want to see whether muscle-specific deletion of S6K1 in mice is able to retain the beneficial effects and eliminate the negative effects experienced by mice deficient for S6K1. Thus, my research focuses mainly on analysing the effect of S6K1mKO on insulin signalling pathway. I am interested in understanding whether S6K1mKO is able to rescue age-induced and diet-induced insulin resistance. As such, I run western blot to analyse the changes in the activity of different proteins involved in the insulin signalling pathway as a result of the knock out of S6K1 specifically in muscle. I am also going to do behavioural assays on these mice to test whether they do have healthier muscles with better running performance and endurance.

TAY Chang Feng Benny

FYP Student, Republic Polytechnic

(September 2017 – January 2018)

Project Title: Protein Homeostasis in aging tissues

Project Description: The mammalian target of rapamycin complex 1 (mTORC1) kinase pathway is studied to be able to regulate lifespan, cell proliferation and link itself to aging-related diseases. It has been observed in older mice, to have an increase in mTORC1s phosphorylation for 4E-binding protein 1 (4E-BP1) and S6 kinase 1 (S6K1) which results in increased cell proliferation. However, it is not studied further to observe the changes to 4E-BP1’s downstream substrate, eukaryotic translation initiation factor 4E (eIF4E) when it comes to aging. In my project, I will study the level of difference in the formation of the translation initiation complex between young and aging mice by pulling down m7-GTP. At the same time, I will also pull down puromycin to study the global translation in aging mice. This pull down will observe if the increased amount of junk proteins being synthesized has any relations with aging.

TAY See Lin

FYP Student, Republic Polytechnic

(February 2018 - August 2018)

Project Title: Investigating the function of muscle ribosomal protein S6 Kinase in the regulation of insulin sensitivity in peripheral metabolic organs

Project Description: Many signalling events occur in the body. In this project, my objective is to analyse the effects of S6K1 knockout in muscle tissue of mice on the cell-to-cell communication between liver and adipose tissue. According to studies , activation of S6K1 has increase protein translation as well as lipid synthesis. In my experiment I would be analyzing liver tissues and adipose tissues from both wild type mice and S6K1mKO mice. The mice would be either be on a HFD or on a NCD, under either fasting condition or injected with insulin before being euthanized for tissue harvesting.This would allow me to study on age-related metabolic diseases such as diabetes type II and insulin resistance as Akt-mTORC1-S6K1 signalling pathway is the main nutrient signalling pathway.I am also interested in to find out how the S6K1 Knockout in muscle tissues would affect lipid metabolism and metabolic activities in the liver through molecular analysis by probing for their relevant signalling components.

TEO Jia Ying

Internship Student, Temasek Polytechnic

(August 2018 - January 2019)

Project Title: The Impact of Muscle Ageing on the Reproduction of Female Mice

Project Description: Female reproductivity and systemic metabolism are tightly connected and reciprocally regulated. Studies have shown that skeletal muscle is crucial for the regulation of systemic metabolism and is one of the major sites for insulin-dependent glucose metabolism. The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that coordinates eukaryotic cell growth and metabolism. Suppression of mTOR signaling either by genetics or pharmaceutical approaches has shown beneficial effects on health such as protection against age-related obesity and locomotion decline in mice. In contrast, uncontrolled mTOR signaling causes early-onset of sarcopenia (loss of muscle mass) and shortens lifespan. Since mTOR signaling is important for regulation of systemic metabolism and muscle aging, I will further investigate whether genetic modification of mTORC1 signaling in skeletal muscle will have any effects on reproductive aging in female mice. I hypothesize that the muscle transgenic mice which have improved muscle healthiness will also extend reproductive lifespan. A possible explanation behind this mechanism would be that myokines secreted from skeletal muscle are protecting the ovaries from aging-associating stresses. Therefore, in this experiment, various muscle parts will be harvested from both control and mutant mice. From the muscle tissues, myoblasts will be isolated and cultured. Myokines secreted from the myoblasts will then be identified and quantified to rule out the potential biomarkers that might affect the regulation of females’ fertility.

LOH Dai Ling Bertina

FYP Student, Republic Polytechnic

(August 2018 - January 2019)

Project Title: Understanding the role of muscle creatine kinase (Ckmm)-Cre in tissue-specific gene expression in transgenic mouse models

Project Description: In a current mouse model of accelerated ageing, genetic knock out of TSC1 gene in muscle (TSC1mKO) leads to increased mTORC1 activity and has shown to cause early-onset sarcopenia and whole-body metabolic perturbations. Genetic knockout utilises the Cre-LoxP system, using Ckmm-Cre, which is known to cause Cre recombination in skeletal muscle and heart. However, it is not known of whether the sarcopenic phenotype of the TSC1mKO mouse is attributable to TSC1 knockout in muscle only or whether knockout in heart also plays a role. Thus, my project focusses on studying the altered mTORC1 signalling activities in the heart muscle following Ckmm-Cre genetic modification.

Andrew S/O AMALADASS

Research Assistant

(May 2017 - April 2019)

Project Description: Andrew is investigating how the muscle knock out of s6k1 (a downstream of the mTOR pathway) in a mice affects its locomotion, strength and cognition. Also, Andrew is the primary person overlooking all the mice in the vivarium and performs PCR to ascertain its genotype.

NG Chong Yi

UROPS Student, NUS Life Sciences

(December 2018 - April 2019)

Project Title: Investigating the effect of S6K1mKO on age-related changes in skeletal muscle morphology

Project Description: S6K1 was thought as a major factor for the growth in the skeletal muscle. The S6K1 whole body knockout mice are lean due to the restriction of muscle cell growth. We have generated skeletal muscle-specific knockout of S6K1 (referred to S6K1mKO mice) to further study the role of S6K1 in muscle function. However, we found that S6K1mKO mice have comparable muscle mass and muscle strength. Interestingly, aging female S6K1mKO mice have better exercise performance and maintain muscle mass compared to control mice. My research will investigate how S6K1 contributing to age-related decline in muscles using immunohistochemistry.