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Mitochondrial and synaptic dysfunction are pathological features of brain ageing and cognitive decline. Synaptic mitochondria are vital for meeting the high energy demands of synaptic transmission. However, little is known about the link between age-related metabolic changes and the integrity of synaptic mitochondria. To this end, we investigated the mechanisms of advanced glycation end product (AGE)-mediated mitochondrial and synaptic stress and evaluated the strategies to eliminate these toxic metabolites. Using aged brain and novel transgenic mice overexpressing neuronal glyoxalase 1 (GLO1), we comprehensively analysed alterations in accumulation/build-up of AGEs and related metabolites in synaptic mitochondria and the association of AGE levels with mitochondrial function. We demonstrated for the first time that synaptic mitochondria are an early and major target of AGEs and the related toxic metabolite methylglyoxal (MG), a precursor of AGEs. MG/AGE-insulted synaptic mitochondria exhibit deterioration of mitochondrial and synaptic function. Such accumulation of MG/AGEs positively correlated with mitochondrial perturbation and oxidative stress in ageing brain. Importantly, clearance of AGE-related metabolites by enhancing neuronal GLO1, a key enzyme for detoxification of AGEs, reduces synaptic mitochondrial AGE accumulation and improves mitochondrial and cognitive function in ageing and AGE-challenged mice. Furthermore, we evaluated the direct effect of AGEs on synaptic function in hippocampal neurons in live brain slices as an ex vivo model and in vitro cultured hippocampal neurons by recording long-term potentiation (LTP) and measuring spontaneously occurring miniature excitatory postsynaptic currents (mEPSCs). Neuronal GLO1 rescues deficits in AGE-induced synaptic plasticity and transmission by full recovery of decline in LTP or frequency of mEPSC. These studies explored crosstalk between synaptic mitochondrial dysfunction and age-related metabolic changes relevant to brain ageing and cognitive decline. Synaptic mitochondria are particularly susceptible to AGE-induced damage, highlighting the central importance of synaptic mitochondrial dysfunction in synaptic degeneration in age-related cognitive decline. Thus, augmenting GLO1 function to scavenge toxic metabolites represents a therapeutic approach to reduce age-related AGE accumulation and improve mitochondrial function and learning and memory. [ABSTRACT FROM AUTHOR]

Glyoxalase 1 (Glo I), a crucial enzyme with multifaceted roles in various physiological and pathological processes, has garnered significant attention in recent years. As a critical detoxification enzyme, Glo I plays a vital role in neutralizing methylglyoxal (MG), a highly reactive and toxic dicarbonyl compound that can irreversibly modify proteins, lipids, and nucleic acids, leading to the formation of advanced glycation end-products (AGEs). The assay involves incubating the Glo I enzyme with glutathione and methylglyoxal, forming S-D-lactoylglutathione. This compound is then converted to lactic acid by adding sulfuric acid, which is oxidized to acetaldehyde using copper. Finally, acetaldehyde reacts with p-phenylphenol in sulfuric acid, creating a colored complex that absorbs light at 570 nm. The study also rigorously examines potential interferences from various biochemical substances, demonstrating the robustness of the proposed method. Experiments were conducted using different combinations of interfering biomolecules, including monosaccharides, amino acids, and proteins, to evaluate their impact on Glo I activity. The results indicate that the p-phenylphenol-Glo I method maintains its effectiveness in the presence of these substances. Furthermore, the method's accuracy was rigorously assessed through intra-day and inter-day analyses, with results expressed as percentage relative error (RE% < 0.4%) to ensure precision in Glo I activity measurements. The current method exhibited excellent linearity across a broad range of 0.005-100 µM Lactate, with a high Pearson's correlation coefficient of 0.989. This work contributes valuable insights into the quantification of Glo I and holds significant implications for research in metabolic disorders and related fields. [ABSTRACT FROM AUTHOR]