P2.48. A BIOORTHOGONAL PUTRESCINE PROBE TO TRACK POLYAMINE UPTAKE AND FLUX UNDER NEUROTOXIC STRESS
Aleksandra Owczarek1, Aleksandra Duchnowska1,2, Aleksandra Skweres1,2, Martyna Nalepa1, Anita Florkowska3, Filip Suchożebski1, Alicja Mąka1, Michał Węgrzynowicz1
1 Mossakowski Medical Research Institute, Polish Academy of Sciences, Laboratory of Molecular Basis of Neurodegeneration, Pawińskiego 5, 02-106, Warsaw, Poland
2 Centre of Postgraduate Medical Education, Doctoral School of Translational Medicine, Marymoncka 99/103, 01-813 Warsaw, Poland
3 International Institute of Molecular Mechanisms and Machines, Polish Academy of Sciences, Laboratory of Stem Cell RNA Metabolism, Flisa 6, 02-247 Warsaw, Poland
INTRODUCTION: Putrescine (Put), like other polyamines, participates in numerous cellular processes in the brain, including neurogenesis, synaptic transmission, plasticity, and response to neurotoxic stress. The cellular specificity of polyamine transport and its stress-induced dynamics remain poorly understood, largely due to the lack of suitable analytical tools.
AIM(S): We aimed to investigate the cellular specificity of Put uptake by comparing neurons and astrocytes. Additionally, we aimed to compare Put accumulation across hippocampal subregions and examine changes in its distribution under hypoxic and excitotoxic stress.
METHOD(S): We employed both an in vitro (mixed cortical neuron/astrocyte primary mouse cultures) and ex vivo (acute mouse hippocampal slices) models. A propargyl-modified Put analogue (TvS-Put) served as a bioorthogonal probe that, upon cellular uptake, was fluorescently tagged via a click chemistry reaction. Hypoxia and excitotoxicity were pharmacologically modelled by exposing slices to CoCl2 and NMDA, respectively.
RESULTS: TvS-Put accumulation in cells was inhibited by non-modified Put and specific polyamine transporter inhibitor demonstrating the probe specificity. The probe was preferentially taken up by neurons over astrocytes in both culture and slice models. In neurons, TvS-Put accumulated primarily in nuclei, followed by somata. In hippocampal slices, the probe accumulated more in CA1 than CA2/3; however this pattern was altered following CoCl₂ or NMDA treatment, with TvS-Put accumulating equally in CA1 and CA2/3, indicating region-specific regulation of Put transport dynamics in response to neurotoxic stress.
CONCLUSIONS: Our findings demonstrate that TvS-Put is a reliable tool for investigating Put transport and distribution in brain cells. This approach opens new avenues for exploring the role of polyamines in the physiology and pathology of the central nervous system.
FINANCIAL SUPPORT: National Science Centre, Poland: 2023/07/X/NZ4/00420