ABSTRACT
Lead (Pb2+), a ubiquitously present heavy metal toxin, has various detrimental effects on memory and cognition. However, the molecular processes affected by Pb2+ causing structural and functional anomalies are still unclear. To explore this, we employed behavioral and proteomic approaches using rat pups exposed to lead acetate through maternal lactation from postnatal day 0 (P0) until weaning. Behavioral results from three-month-old rats clearly emphasized the early life Pb2+ exposure induced impairments in spatial cognition. Further, proteomic analysis of synaptosomal fractions revealed differential alteration of 289 proteins, which shows functional significance in elucidating Pb2+ induced physiological changes. Focusing on the association of Small Ubiquitin-like MOdifier (SUMO), a post-translational modification, with Pb2+ induced cognitive abnormalities, we identified 45 key SUMO target proteins. The significant downregulation of SUMO target proteins such as metabotropic glutamate receptor 3 (GRM3), glutamate receptor isoforms 2 and 3 (GRIA 2 and GRIA3) and flotilin-1 (FLOT1) indicates SUMOylation at the synapses could contribute to and drive Pb2+ induced physiological imbalance. These findings identify SUMOylation as a vital protein modifier with potential roles in hippocampal memory consolidation and regulation of cognition. Data availbility: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD034212".
ABSTRACT
Mitochondrial membrane potential (Ψm) is a critical parameter that can be used to determine cellular well-being. As it is a direct measure of the cell's ATP generating capability, in recent years, this key component in cell biology has been the subject of thousands of biochemical and biophysical investigations. Membrane-permeant fluorescent dyes, like tetramethylrhodamine ethyl ester (TMRE), have been predominantly employed to monitor ΔΨm in cells. These dyes are typically lipophilic cationic compounds that equilibrate across membranes in a Nernstian fashion, thus accumulating into the mitochondrial membrane matrix space in inverse proportion to Ψm. However, the bath loading method practiced for labelling tissue slices with these cationic dyes poses limitations in the form of non-specificity and low signal to noise ratio, which compromises the precision of the results. Therefore, we introduce an alternative way for TMRE loading to image the ΔΨm in tissue slices by utilizing a low resistance glass pipette attached to a pressure injector. This method shows highly precise fluorescent dye labelling of the mitochondria and offers maximum output intensity, in turn enhancing signal to noise ratio.
