Quantitative Proteomics of Synaptic and Nonsynaptic Mitochondria: Insights for Synaptic Mitochondrial Vulnerability

Synaptic mitochondria are essential
for maintaining calcium homeostasis
and producing ATP, processes vital for neuronal integrity and synaptic
transmission. Synaptic mitochondria exhibit increased oxidative damage
during aging and are more vulnerable to calcium insult than nonsynaptic
mitochondria. Why synaptic mitochondria are specifically more susceptible
to cumulative damage remains to be determined. In this study, the
generation of a super-SILAC mix that served as an appropriate internal
standard for mouse brain mitochondria mass spectrometry based analysis
allowed for the quantification of the proteomic differences between
synaptic and nonsynaptic mitochondria isolated from 10-month-old mice.
We identified a total of 2260 common proteins between synaptic and
nonsynaptic mitochondria of which 1629 were annotated as mitochondrial.
Quantitative proteomic analysis of the proteins common between synaptic
and nonsynaptic mitochondria revealed significant differential expression
of 522 proteins involved in several pathways including oxidative phosphorylation,
mitochondrial fission/fusion, calcium transport, and mitochondrial
DNA replication and maintenance. In comparison to nonsynaptic mitochondria,
synaptic mitochondria exhibited increased age-associated mitochondrial
DNA deletions and decreased bioenergetic function. These findings
provide insights into synaptic mitochondrial susceptibility to damage.

突触线粒体（Synaptic mitochondria）是维持钙稳态（calcium homeostasis）与生成三磷酸腺苷（ATP）的核心结构，这两类过程对于维持神经元完整性与突触传递具有关键作用。突触线粒体在衰老过程中氧化损伤程度更高，且相较于非突触线粒体（nonsynaptic mitochondria），其更易受到钙损伤的影响。为何突触线粒体会特异性地更易遭受累积性损伤，这一问题仍有待阐明。本研究中，我们构建了超SILAC（super-SILAC）混合体系，该体系可作为小鼠脑线粒体质谱（mass spectrometry）分析的适宜内标，实现了对10月龄小鼠分离获得的突触线粒体与非突触线粒体之间蛋白质组学差异的定量分析。我们共鉴定出突触与非突触线粒体共有的2260种蛋白质，其中1629种被注释为线粒体相关蛋白。对两类线粒体共有的蛋白质进行定量蛋白质组学分析后发现，共有522种蛋白质的表达存在显著差异，这些蛋白质参与氧化磷酸化、线粒体分裂/融合、钙转运以及线粒体DNA复制与维持等多条通路。与非突触线粒体相比，突触线粒体的年龄相关性线粒体DNA缺失更为显著，且其生物能学功能有所下降。本研究结果为理解突触线粒体易受损伤的机制提供了新的见解。