With the use of advanced microscopy techniques, researchers were able to show how a natural polyamine, spermidine, could enhance treatment strategies against neurodegeneration associated with Alzheimer’s disease.
During her PhD, Dr Dumisile Lumkwana, also a former analyst at CAF Fluorescence Microscopy Unit, made use of superresolution microscopy and correlative light and electron microscopy (CLEM) to study the effects of spermidine in neuronal cells in which Alzheimer’s-related effects had been induced. This work was recently published in the journal Frontiers in Cell and Developmental Biology (DOI:10.3389/fcell.2022.819571).
Models for Alzheimer’s disease
During Alzheimer’s disease, certain proteins accumulate and aggregate in neuronal cells, causing neurotoxicity. One such protein is amyloid precursor protein (APP), which in turn leads to the generation of the aggregate-prone peptide amyloid-beta. Using cells genetically modified to produce excessive levels of APP and amyloid-beta, its accumulation mimics the protein accumulation associated with Alzheimer’s disease. Some environmental pollutants and agricultural pesticides, such as paraquat, are neurotoxic and have also been associated with neurodegenerative disease.
Under supervision of Prof Ben Loos from the Department of Physiological Sciences, Dr Lumkwana investigated the efficacy of spermidine 1) in cells overexpressing APP, 2) in cells treated with paraquat and 3) in a mouse model where neuronal damage had been induced by paraquat administration.
Cellular viability was reduced over time in mutant neuronal cells overexpressing APP, while spermidine improved the cellular viability over time significantly. Using flow cytometry, performed by Dr Rozanne Adams, it was also shown that paraquat reduced neuronal cell viability and induced a significant increase in production of reactive oxygen species (ROS) in the cells. Both low and high concentrations of spermidine improved cell viability significantly, but ROS production was only reduced when a low concentration of spermidine was used, suggesting that the effects of spermidine is concentration dependent and should be investigated accordingly. These results showed that the use of both APP overexpressing cells and cells exposed to paraquat toxicity was ideal to study the mechanisms through which spermidine could provide protection against neurodegeneration.
Targeting the cellular clean-up process
One such mechanism of action could be through the cellular process called autophagy, which is responsible for removing protein aggregates, toxic agents and other damaged cell content. During autophagy, cargo collects in vacuoles in the cell and is subsequently taken through a process of degradation. First, molecules are collected in a phagophore, which then develops into an autophagosome and eventually fuses with a lysosome to form what is called an autolysosome, where pH-dependent degradation takes place. The rate of protein degradation through the process is called the autophagic flux, and an increased autophagic flux could be beneficial, especially since autophagy activity has been shown to decline during ageing. Increasing autophagy activity is the focus of many studies to find improved treatment strategies against neurodegenerative diseases, especially those related to ageing.
Assessing the autophagic flux
The LSM780 confocal microscope with ELYRA PS1 superresolution platforms in the CAF Microscopy Unit (previously Fluorescence Microscopy Unit) was used to visualise a key component of matured autophagosomes, namely microtubule-associated protein-1 light chain or LC3 protein. This protein can be used to measure various parameters of the autophagic flux. Here, a labelling strategy whereby LC3 presented yellow in autophagosomes and red in autolysosomes with confocal microscopy revealed that spermidine treatment at high (10 µM) and low (1 µM) concentrations significantly increased the number of autolysosomes but only high concentrations of spermidine increased the number of autophagosomes (Fig.8). The turnover time of the whole autophagic pool was reduced from 2 h in control cells to 0.64 h in the low-concentration treatment group and to 1.21 h in the high-concentration treatment group. These results suggest that there is a concentration-dependent increase in the autophagic flux in response to spermidine.
Correlative microscopy to study the autophagic vacuoles
Using transmission electron microscopy, which provides superior resolution to confocal microscopy, autophagic vacuole sizes were measured and found to be reduced in spermidine treatment groups. However, with electron microscopy, autophagosomes and autolysosomes are indistinguishable (hence referred to only as autophagic vacuoles), which prevents accurate analysis of changes in vacuolar size in the context of the autophagic flux. To distinguish autophagosomes from autolysosomes in the 3D cellular ultrastructure, a CLEM workflow had to be implemented. Structured illumination microscopy (SIM), which improves the resolution of fluorescence imaging, was first used to localise the autophagosomes. At the time of this study, 3D electron microscopy was not available at Stellenbosch University, so samples were fixed, prepared for electron microscopy and then sent to the Electron Microscopy Laboratory in the Science Technology Platform of the Francis Crick Institute in London for acquisition of electron microscopy micrographs in 3D on a focused-ion-beam scanning electron microscope (FIB-SEM). The research team of Prof Loos then used the superresolution microscopy results obtained in CAF to localise specifically the autophagosomes in the electron micrographs obtained in the United Kingdom (Fig. 9). After localisation, the autophagosomes had to be manually segmented through the entire stack of electron microscopy images to provide a 3D model of the autophagosomes in their entire volume before morphometric analysis could be performed (Fig. 10). One of the main role players in this process of correlation, segmentation and analysis was Dr Jurgen Kriel, who is now the operator of the ThermoFisher VolumeScope SEM at Tygerberg Medical Campus, but Nicola Heathcote, at the time an honours student and now a PhD student in the Loos lab, also played an important role.
The results of CLEM revealed that spermidine played a significant role in size regulation of the autophagosomes, which is related to effective cargo clearance. The volume and surface area of autophagosomes were increased in the low-concentration treated cells, while these parameters were reduced in the high-concentration treatment group. These results confirmed the concentration-dependent effect of spermidine, which deserves further study, and further highlighted the requirement for high-precision identification of cellular organelles in electron micrographs through the identification strategies made possible with fluorescence methods and subsequent correlation.
Effects of spermidine on the cellular transport proteins
Autophagosome transport relies on the transport systems of the cell to accommodate association with lysosomes for fusion. Tubulin, especially in an acetylated state, is one of the scaffold proteins providing a transport mode for organelles such as autophagosomes and is structurally most effectively studied with superresolution techniques due to its fine structure. Using both superresolution microscopy platforms, namely SIM and stochastic optical reconstruction microscopy (STORM), on the neuronal cell models, it was observed that tubulin signal and density increased in paraquat-affected cells treated with spermidine (Fig. 11). However, from measurements in mouse brains, it seemed that the effects of spermidine and paraquat treatments on acetylated tubulin levels were region specific. The localisation and structure of acetylated tubulin were not determined in the tissue samples, but the results obtained in the cells are motivation for further study on the effects of spermidine on transport proteins in live models.
Accurate measurements of amyloid precursor protein accumulation and clearance
Visualisation and accurate quantification of protein accumulation and clearance provide strong evidence of the efficacy of therapeutic drugs for neurodegenerative diseases. Here, the STORM superresolution method was used to determine the number of APP aggregates as well as their relative sizes. With conventional confocal microscopy, structures smaller than 200 nm cannot be measured accurately, while SIM allows only accurate measurements down to 100 nm. With STORM, resolution can be improved to several nanometres. For this method, samples are usually visualised in a buffer that allows the fluorescence molecules to blink rather than constantly emit their fluorescence together with neighbouring fluorophores. The blinking effect enables the researcher, through software algorithms, to localise the single fluorescence molecules used for protein labelling to subresolution precision and to reconstitute the fluorescence image from the map of localised fluorophores instead of their actual emission.
APP overexpression was induced by administration of butyric acid (BA) for 24 h and 48 h respectively. APP clusters ranged from 6 to 50 nm2, with an abundance of APP clusters from 6 to 10 nm2 at 24 h. The number of APP clusters at the 48-h timepoint was significantly increased for all sizes. In the presence of spermidine, clearance of APP clusters was evident, especially in the reduced number of clusters 6-25 nm2 at the 24-h timepoint, while APP cluster clearance of all sizes was evident at 48 h (Fig.12). This study clearly shows visually how treatment with spermidine reduces protein accumulation and mediates clearance of such aggregates, revealing its potential in the treatment and prevention of diseases such as Alzheimer’s disease.
Advanced microscopy techniques available for all
The correlative work of Dr Lumkwana was the forerunner for the acquisition of ThermoFisher Apreo Volumescope SEM for Stellenbosch University. Now housed by the CAF at the Tygerberg Medical Campus, the Volumescope is operated by Dr Kriel who collaborates closely with the team of the Electron Microscopy Laboratory of the Francis Crick Institute to ensure that methods are adequately implemented here. The availability of volumetric electron microscopy at Stellenbosch University opens opportunities for researchers in South Africa to study subcellular structure at much higher precision than what was previously possible.
The application of superresolution techniques has not been adopted in South Africa to the same extent as in areas such as Europe and the United States of America. While SIM does not require specialised sample preparation, only optimal signal from fluorescent markers, STORM requires an optimised environment for the fluorophores to blink. This has proven to be quite challenging, limiting the use of STORM. This study showcases how superresolution microscopy, in correlation with electron microscopy, can not only reveal novel information but also provide a means for structural measurements of much higher precision than confocal microscopy.