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. 2022 May 17:10:819571.
doi: 10.3389/fcell.2022.819571. eCollection 2022.

Investigating the Role of Spermidine in a Model System of Alzheimer's Disease Using Correlative Microscopy and Super-resolution Techniques

D Lumkwana  1 , C Peddie  2 , J Kriel  3 , L L Michie  4 , N Heathcote  4 , L Collinson  2 , C Kinnear  5 , B Loos  4
Affiliations

Investigating the Role of Spermidine in a Model System of Alzheimer's Disease Using Correlative Microscopy and Super-resolution Techniques

D Lumkwana et al. Front Cell Dev Biol. .

Abstract

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 of a high concentration of spermidine on autophagic flux, neuronal toxicity, and protein clearance in in vitro models of paraquat (PQ) induced neuronal toxicity and amyloid precursor protein (APP) overexpression, 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 Microscopy (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 overexpression. In the in vivo scenario, we demonstrate brain region-specific susceptibility to PQ-induced neuronal toxicity, with the hippocampus being highly susceptible compared to 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.

Keywords: Alzheimer’s disease; autophagy; correlative light and electron microscopy; direct stochastic optical reconstruction microscopy; focused ion beam scanning electron microscopy; spermidine; super-resolution structured illumination.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Spermidine induces autophagic flux and improves cellular viability in a concentration-dependent manner. (A) densitometric quantification and (B) representative western blot for LC3-II protein levels upon autophagy induction using three different concentrations (0.1, 1 and 10 μM) of spermidine in the presence and absence saturating concentrations of bafilomycin. Data are presented as mean ± SEM, n = 3. * = p < 0.05 vs. control, $ = p < 0.05 vs. 0.1 μM Spd + BafA1 and % = p < 0.05 vs. 10 μM Spd, arbitrary units (a.u). (C) LC3-II turnover revealing a concentration-dependent flux by spermidine. * = p < 0.05 vs. control, # = p < 0.05 vs. 0.1 μM Spd and $ = p < 0.05 vs. 1 μM Spd. (D) Reductive capacity in GT1-7 cells following treatment with three different concentrations of spermidine. Data are presented as mean ± SEM, n = 3 with 6 replicates per group. * = p < 0.05 vs. control, # = p < 0.05 vs. 0.1 μM Spd and $ = p < 0.05 vs. 1 μM Spd.
FIGURE 2
FIGURE 2
Spermidine induces autophagic flux in a concentration-dependent manner. (A,B) quantitative analysis and (C) representative fluorescence micrographs of autophagosome pool size (A,C) and autolysosome pool size (B,C) in GT1-7 cells following treatment with 1 and 10 μM spermidine in the presence and absence of saturating concentrations bafilomycin. Data are presented as mean ± SEM, n = 3, with a total of 30 cells analysed per treatment group. * = p < 0.05 vs control, # = p < 0.05 vs BafA1, @ = p < 0.05 vs 1 μM Spd and % = p < 0.05 vs 10 μM Spd, arbitrary units (a.u). Scale bar: 5 μm. (D) Autophagosome flux under basal and induced conditions calculated from the initial slope of autophagosomes accumulation. Autophagosome flux increased in a concentration-depended manner, raising from 2.0 autophagosomes/h/cell under basal levels to 3.1 autophagosomes/h/cell and 3.3 autophagosomes/h/cell under induced conditions with 1 and 10 μM Spd respectively.
FIGURE 3
FIGURE 3
Spermidine affects the size of AVs in a concentration-dependent manner but has no effect on the number of AVs. (A,B) morphometric analysis and (C) representative TEM micrographs of autophagic vacuoles (AVs) in GT1-7 cells following treatment with 1 and 10 μM spermidine in the presence and absence of saturating concentrations bafilomycin. Data are presented as mean ± SEM, n = 2 with a total of 30 images analysed per treatment group. * = p < 0.05 vs control, # = p < 0.05 vs. BafA1, $ = p < 0.05 vs. 1 μM Spd + BafA1, @ = p < 0.05 vs. 1 μM Spd and % = p < 0.05 vs. 10 μM Spd, arbitrary units (a.u). Arrowheads indicate AVs. Scale bar: 2000 nm.
FIGURE 4
FIGURE 4
Spermidine induces autophagy. (A–F) Three colour micrograph of SR-SIM, FIB-SEM and overlay of SR-SIM and FIB-SEM showing the accumulation of GFP-LC3 and RFP-LC3ΔG positive structures following treatments. (C,F) complex structures of high electron density. (Ci) region of interest showing GFP-LC3 and RFP-LC3ΔG positive structures decorating conglomerate structure. (Fi) region of interest showing the localisation of RFP-LC3ΔG positive structures inside the conglomerate structure as part of cytoplasmic cargo. Scale bar: 1 μm.
FIGURE 5
FIGURE 5
Differential response in cargo selection upon treatment. (A) Selective region of interest indicating autophagosome (white arrow heads) in FIB-SEM (Left), SR-SIM (middle) and CLEM (right) for the control (untreated) group, 1 μM Spd, 1 μM Spd + BafA1, 10 μM Spd and 10 μM Spd + BafA1. (B) FIB-SEM micrograph showing distinct cargo contained within the autophagosome (white arrow heads) following treatments. 10 μM Spd + BafA1 treated group recruits cargo characterised by the abundance of small intra-autophagosomal vesicle-like structures of homogeneous electron density. Scale bar: 1 μm and 200 nm.
FIGURE 6
FIGURE 6
Effect of spermidine on cargo clearance and size regulation. (A) Orthogonal views of FIB-SEM stack showing segmented autophagosomes (scale bar: 1 μm). (B) Morphometric analysis of autophagosome volume and (C) surface area following treatments. Data are presented as mean ± SEM. A total of 15–25 autophagosomes analysed per treatment group. Spermidine at low concentration induces autophagosomes capable of large volume clearance. * = p < 0.05 vs. control, # = p < 0.05 vs. BafA1, @ = p < 0.05 vs. 1 μM Spd and % = p < 0.05 vs. 10 μM Spd.
FIGURE 7
FIGURE 7
Low concentration of spermidine reduces ROS production and delay cell death onset following paraquat-induced neurotoxicity. (A) Reductive capacity indicating an improvement in cellular viability in cells pre-treated with spermidine compared to PQ alone. Data are presented as mean ± SEM, n = 3 with 6 replicates per group. * = p < 0.05 vs control, # = p < 0.05 vs PQ. (B) quantitative analysis of mitochondrial ROS production and (C) representative fluorescence micrographs following treatments. MitoSox fluorescence intensity was increased in PQ treated group, while decreased with 1 μM Spd + PQ. Scale bar: 20 μm. (D,E) quantitative analysis of cell death onset following treatments. PI positive cells were reduced in 1 μM Spd + PQ compared to PQ treated group. Data are presented as mean ± SEM, n = 3 with 2 replicates per group. * = p < 0.05 vs control group, # = p < 0.05 vs. PQ, @ = p < 0.05 vs. 1 μM Spd, % = p < 0.05 vs. 10 μM Spd, and $ = p < 0.05 vs 1 μM Spd + PQ.
FIGURE 8
FIGURE 8
Spermidine increases the acetylation signal of tubulin in paraquat treated cells. (A) densitometric quantification and (B) representative western blot for acetylated α-tubulin protein levels in GT1-7 cells following treatment with a low and high concentration of spermidine followed by exposure to paraquat. Data are presented as mean ± SEM, n = 3. (C,D) representative micrographs for acetylated α-tubulin with SR-SIM (scale bar: 5 and 1 μm) and dSTORM (scale bar: 2 and 0.5 μm) showing highest intensity signal with 1 μM Spd + PQ compared to PQ. Arrowheads indicate regions of strong acetylated α-tubulin signal.
FIGURE 9
FIGURE 9
Spermidine protects against APP-induced cytotoxicity and reduces the size and number of APP clusters. (A) Reductive capacity and (B) western blot analysis of APP, LC3-I/II and LAMP2A protein levels following treatment with a low and high concentration of spermidine in N2aSwe cells treated with BA overtime to overexpress APP (n = 3). * = p < 0.05 vs. control, # = p < 0.05 vs. 24 h BA and $ = p < 0.05 vs. 48 h BA. (C–E) Size distribution of APP clusters and representative dSTORM micrographs in Gauss mode showing the localisation of APP clusters. Spermidine reduces the size and number of APP clusters. Scale bar: 0.5 μm.
FIGURE 10
FIGURE 10
Spermidine reduces lipid peroxidation and amyloidogenic processing in the hippocampus and cortex region. Representative western blot and densitometric analysis of 4HNE adducts (35, 45 and 52 kDa) and APP protein levels in the hippocampus (A and B) and (C and D) cortex. The data are expressed as mean ± SEM. A total of 6–10 animals were used per group (n = 6–10). Spermidine in combination with PQ reduced 4HNE and APP protein levels compared to PQ treated group. * = p < 0.05 vs. control, # = p < 0.05 vs. PQ and @ = p < 0.05 vs. 0.3 mM Spd, arbitrary units (a.u).
FIGURE 11
FIGURE 11
Differential effect of spermidine on tubulin acetylation and autophagic activity in the hippocampus and cortex. (A,B) Representative western blot and densitometric analysis of acetylated α-tubulin and p62 protein levels in the hippocampus and (C,D) cortex. The data are expressed as mean ± SEM. A total of 6–10 animals were used per group (n = 6–10). Spermidine in combination with PQ increased acetylated α-tubulin and p62 compared to PQ treated group in the hippocampus. In the cortex region, combination treatment of the low concentration spermidine increased p62 protein levels, while co-treatment with the high concentration of spermidine decreased acetylated α-tubulin with no effect on p62 levels. * = p < 0.05 vs. control, # = p < 0.05 vs. PQ, @ = p < 0.05 vs. 0.3 mM Spd, $ = p < 0.05 vs. 0.3 mM Spd + PQ and % = p < 0.05 vs. 3 mM Spd, arbitrary units (a.u).
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