Aging-induced trnaGlu-derived fragments disrupt glutamate biosynthesis by targeting mitochondrial translation-dependent cristae organization

Academic Background

Mitochondrial cristae are inward protrusions of the inner mitochondrial membrane that undergo significant morphological changes during aging. However, the molecular mechanisms leading to these changes and their contribution to brain aging remain unclear. Maintaining the ultrastructure of mitochondrial cristae is crucial for the normal function of respiratory enzymes present in the cristae. If the cristae structure is disrupted, the enzyme activity on the inner membrane will be significantly reduced. Glutaminase (GLS) is an important enzyme located in the cristae responsible for catalyzing the production of glutamate and maintaining glutamate levels in the neuronal internal environment. As the most abundant neuroactive amino acid and major excitatory neurotransmitter, glutamate levels gradually decline during brain aging, often accompanied by memory decline.

Transfer RNA-derived small RNAs (tsRNAs) are produced from mature tRNAs under various stresses and participate in multiple physiological and pathological processes. However, the functions and molecular mechanisms of tsRNAs in organ specificity have not been fully elucidated. This study investigated the role and mechanism of a nuclear-encoded tRNA Glu-CTC (tRNA Glu) derived tsRNA, namely glu-50tsRNA-CTC, in brain aging.

Research Source

This paper was authored by researchers including Dingfeng Li, Xinyi Gao, Xiaolin Ma, Ming Wang, Chuandong Cheng, Tian Xue, Feng Gao, Yong Shen, Juan Zhang, and Qiang Liu from the University of Science and Technology of China. The study was published in the journal Cell Metabolism on May 7, 2024.

Detailed Research Introduction

Experimental Design and Process

This study analyzed the expression patterns of tsRNAs in the medial prefrontal cortex (mPFCs) of 3-month-old and 22-month-old mice through small RNA sequencing (RNA-seq), finding that glu-50tsRNA-CTC was highly increased. Northern blot was used to identify and validate the accumulation of glu-50tsRNA-CTC in aged mouse mPFCs, and in situ hybridization combined with immunofluorescence experiments confirmed its specific accumulation in glutamatergic neurons.

The study further revealed the generation mechanism of glu-50tsRNA-CTC. Through RNA pull-down and immunoprecipitation (IP), it was found that glu-50tsRNA-CTC is imported into mitochondria by binding to leucyl-tRNA synthetase 2 (LARS2), where it competitively inhibits the aminoacylation of mt-tRNALeu, thereby impairing mitochondrial protein synthesis and ultimately leading to cristae damage and decreased GLS levels.

Using transmission electron microscopy (TEM) and quantitative PCR (qPCR), the study also found that attenuating glu-50tsRNA-CTC could alleviate cristae defects and glutamate level decline in the mPFCs of aged mice, significantly improving their synaptic structure and memory ability.

Main Research Findings

Aging-induced generation of glu-50tsRNA-CTC in the brain

Through RNA-seq analysis, it was discovered that tsRNA glu-50tsRNA-CTC is formed by specific cleavage of mature tRNA Glu-CTC by angiogenin (Ang) and significantly accumulates in the mPFCs of aged mice. Further experiments showed that dephosphorylation of Ang induces its nucleocytoplasmic translocation, leading to the generation and accumulation of glu-50tsRNA-CTC in the cytoplasm.

Accumulation of glu-50tsRNA-CTC in mitochondria of glutamatergic neurons

The study found that glu-50tsRNA-CTC specifically accumulates in the mitochondria of glutamatergic neurons, achieved through a LARS2-mediated import mechanism. RNA pull-down and immunoprecipitation experiments verified the interaction between glu-50tsRNA-CTC and LARS2, finding that the uua sequence of glu-50tsRNA-CTC and the anticodon domain of LARS2 are key to their interaction.

glu-50tsRNA-CTC interferes with mt-tRNALeu binding to LARS2 and disrupts mt-tRNALeu aminoacylation

By increasing the expression of glu-50tsRNA-CTC, a significant reduction in LARS2-bound mt-tRNALeu was observed, indicating that glu-50tsRNA-CTC competes with mt-tRNALeu for binding to LARS2, leading to impaired mt-tRNALeu aminoacylation and subsequent disruption of mitochondrial protein translation.

glu-50tsRNA-CTC-mediated mitochondrial translation defects lead to mitochondrial cristae damage

The study verified that overexpression of glu-50tsRNA-CTC or LARS2 inhibition results in mitochondrial protein translation defects, subsequently disrupting normal mitochondrial cristae organization, leading to significantly reduced cristae efficiency and increased cristae diameter. Conversely, LARS2 overexpression was found to alleviate these defects, restoring mt-tRNALeu aminoacylation and mitochondrial translation.

Normal mitochondrial cristae organization maintains GLS-dependent glutamate synthesis

The study found that glu-50tsRNA-CTC reduces glutamate levels in mitochondria by damaging mitochondrial cristae organization and GLS protein stability. TEM observation and quantitative analysis revealed that glu-50tsRNA-CTC can lead to GLS ubiquitination and degradation, further reducing glutamate levels. The use of proteasome inhibitors could prevent GLS degradation, confirming the involvement of the ubiquitination mechanism.

Reduction of glu-50tsRNA-CTC levels in the brain improves memory in aged mice

Using behavioral experiments (such as Morris water maze, object recognition, and fear conditioning tasks), it was found that interfering with glu-50tsRNA-CTC could significantly improve spatial and contextual memory in aged mice, indicating that glu-50tsRNA-CTC is an important factor causing aging-related memory decline in the brain.

Conclusion and Research Value

By exploring the generation and accumulation of glu-50tsRNA-CTC and its disruption of glutamate biosynthesis through interfering with mitochondrial translation-dependent cristae organization, this study deeply reveals the pathological role of tsRNAs in brain aging and memory decline. Meanwhile, the ASO therapeutic strategy targeting glu-50tsRNA-CTC provides a potential treatment approach for aging-related cognitive dysfunction. This research not only provides important scientific information on the molecular mechanisms of brain aging but also has significant application value for developing new treatment methods.