REL 2022-02-04 2022-06-22 2022-06-22 0000-0002-8329-5419 Christopher J Peddie The Francis Crick Institute 1 Midland Road London United Kingdom NW1 1AT 0000-0002-8329-5419 Christopher J Peddie The Francis Crick Institute 1 Midland Road London United Kingdom NW1 1AT Peddie CJ Collinson LM 140.5 10.6019/EMPIAR-10946 CLEM cell Lumkwana D Peddie CJ Kriel J Michie LL Heathcote N Collinson LM Kinnear C Loos B Frontiers in cell and developmental biology Front Cell Dev Biol 2022 English 10.3389/fcell.2022.819571

Background: Spermidine has recently received major attention for its potential therapeutic benefits in the context of neurodegeneration, cancer, and aging. However, it is unclear whether concentration dependencies of spermidine exist, to differentially enhance autophagic flux. Moreover, the relationship between low or high autophagy activity relative to basal neuronal autophagy flux and subsequent protein clearance as well as cellular toxicity has remained largely unclear. Methods: Here, we used high resolution imaging and biochemical techniques to investigate the effects of a low and high concentration of spermidine on autophagic flux, neuronal toxicity, and protein clearance in models of paraquat (PQ) induced neuronal toxicity and APP over-expression, as well as in an in vivo model of PQ-induced rodent brain injury. Results: Our results reveal that spermidine induces autophagic flux in a concentration-dependent manner, however the detectable change in the autophagy response critically depends on the specificity and sensitivity of the method employed. By using correlative imaging techniques through Super-Resolution Structured Illumination (SR-SIM) and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM), we demonstrate that spermidine at a low concentration induces autophagosome formation capable of large volume clearance. In addition, we provide evidence of distinct, context-dependent protective roles of spermidine in models of Alzheimer’s disease. In an in-vitro environment, a low concentration of spermidine protected against PQ-induced toxicity, while both low and high concentrations provided protection against cytotoxicity induced by APP over-expression. In the in vivo scenario, we demonstrate brain region-specific susceptibility to PQ-induced neuronal toxicity, with the hippocampus being highly susceptible followed by the cortex. Regardless of this, spermidine administered at both low and high dosages protected against paraquat-induced toxicity. Conclusions: Taken together, our results demonstrate that firstly, administration of spermidine may present a favourable therapeutic strategy for the treatment of Alzheimer’s disease and secondly, that concentration and dosage dependent precision autophagy flux screening may be more critical for optimal autophagy and cell death control than previously thought.

S-BSST805 MRC013_EM04295_01_7J_XZ: Control treatment /data micrographs - multiframe TIFF TIFF 851 1 UNSIGNED BYTE 5472 50.0 2894 50.0

MRC013_EM04295_01_7J_XZ: Control treatment Processed FIB-SEM image series that has been reoriented to the XZ view to match the corresponding SR-SIM dataset (BioStudies B-SST805). Images were acquired at a nominal isotropic resolution of 5 nm. The SEM was operated at an accelerating voltage of 1.5 kV with 1 nA current. The EsB detector was used with a grid voltage of 1200 V. Ion beam milling was performed at an accelerating voltage of 30 kV and current of 700 pA. Image processing comprised SIFT registration, denoising (gaussian blur [0.8 pixel radius], smart sharpen [60%, 10 pixel radius, highlights suppressed], smart sharpen [100%, 2 pixel radius, highlight suppressed]), grey level adjustment as required, 8 bit conversion, and rotation and reslice to XZ.

MRC013_EM04295_02_7M_XZ: Bafilomycin treatment /data micrographs - multiframe TIFF TIFF 921 1 UNSIGNED BYTE 5907 50.0 5165 50.0

MRC013_EM04295_02_7M_XZ: Bafilomycin treatment Processed FIB-SEM image series that has been reoriented to the XZ view to match the corresponding SR-SIM dataset (BioStudies B-SST805). Images were acquired at a nominal isotropic resolution of 5 nm. The SEM was operated at an accelerating voltage of 1.5 kV with 1 nA current. The EsB detector was used with a grid voltage of 1200 V. Ion beam milling was performed at an accelerating voltage of 30 kV and current of 700 pA. Image processing comprised SIFT registration, denoising (gaussian blur [0.8 pixel radius], smart sharpen [60%, 10 pixel radius, highlights suppressed], smart sharpen [100%, 2 pixel radius, highlight suppressed]), grey level adjustment as required, 8 bit conversion, and rotation and reslice to XZ.

MRC013_EM04295_03_9M_XZ: 1 micromolar spermidine treatment /data micrographs - multiframe TIFF TIFF 800 1 UNSIGNED BYTE 5000 50.0 5500 50.0

MRC013_EM04295_03_9M_XZ: 1 micromolar spermidine treatment Processed FIB-SEM image series that has been reoriented to the XZ view to match the corresponding SR-SIM dataset (BioStudies B-SST805). Images were acquired at a nominal isotropic resolution of 5 nm. The SEM was operated at an accelerating voltage of 1.5 kV with 1 nA current. The EsB detector was used with a grid voltage of 1200 V. Ion beam milling was performed at an accelerating voltage of 30 kV and current of 700 pA. Image processing comprised SIFT registration, denoising (gaussian blur [0.8 pixel radius], smart sharpen [60%, 10 pixel radius, highlights suppressed], smart sharpen [100%, 2 pixel radius, highlight suppressed]), grey level adjustment as required, 8 bit conversion, and rotation and reslice to XZ.

MRC013_EM04295_04_8O_XZ: 1 micromolar spermidine / bafilomycin treatment /data micrographs - multiframe TIFF TIFF 1001 1 UNSIGNED BYTE 5397 50.0 5349 50.0

MRC013_EM04295_04_8O_XZ: 1 micromolar spermidine / bafilomycin treatment Processed FIB-SEM image series that has been reoriented to the XZ view to match the corresponding SR-SIM dataset (BioStudies B-SST805). Images were acquired at a nominal isotropic resolution of 5 nm. The SEM was operated at an accelerating voltage of 1.5 kV with 1 nA current. The EsB detector was used with a grid voltage of 1200 V. Ion beam milling was performed at an accelerating voltage of 30 kV and current of 700 pA. Image processing comprised SIFT registration, denoising (gaussian blur [0.8 pixel radius], smart sharpen [60%, 10 pixel radius, highlights suppressed], smart sharpen [100%, 2 pixel radius, highlight suppressed]), grey level adjustment as required, 8 bit conversion, and rotation and reslice to XZ.

MRC013_EM04295_05_8Q_XZ: 10 micromolar spermidine treatment /data micrographs - multiframe TIFF TIFF 981 1 UNSIGNED BYTE 3074 50.0 8420 50.0

MRC013_EM04295_05_8Q_XZ: 10 micromolar spermidine treatment Processed FIB-SEM image series that has been reoriented to the XZ view to match the corresponding SR-SIM dataset (BioStudies B-SST805). Images were acquired at a nominal isotropic resolution of 5 nm. The SEM was operated at an accelerating voltage of 1.5 kV with 1 nA current. The EsB detector was used with a grid voltage of 1200 V. Ion beam milling was performed at an accelerating voltage of 30 kV and current of 700 pA. Image processing comprised SIFT registration, denoising (gaussian blur [0.8 pixel radius], smart sharpen [60%, 10 pixel radius, highlights suppressed], smart sharpen [100%, 2 pixel radius, highlight suppressed]), grey level adjustment as required, 8 bit conversion, and rotation and reslice to XZ.

MRC013_EM04295_06_6K_XZ: 10 micromolar spermidine / bafilomycin treatment /data micrographs - multiframe TIFF TIFF 900 1 UNSIGNED BYTE 7052 50.0 5196 50.0

MRC013_EM04295_06_6K_XZ: 10 micromolar spermidine / bafilomycin treatment Processed FIB-SEM image series that has been reoriented to the XZ view to match the corresponding SR-SIM dataset (BioStudies B-SST805). Images were acquired at a nominal isotropic resolution of 5 nm. The SEM was operated at an accelerating voltage of 1.5 kV with 1 nA current. The EsB detector was used with a grid voltage of 1200 V. Ion beam milling was performed at an accelerating voltage of 30 kV and current of 700 pA. Image processing comprised SIFT registration, denoising (gaussian blur [0.8 pixel radius], smart sharpen [60%, 10 pixel radius, highlights suppressed], smart sharpen [100%, 2 pixel radius, highlight suppressed]), grey level adjustment as required, 8 bit conversion, and rotation and reslice to XZ.