Introduction
In recent years, a significant progress occurred concerning the nature of alpha-synuclein (alpha-syn)-related cytopathology, encompassing small protein aggregates and frank neuronal inclusions, which develop within damaged catecholamine neurons. This mostly applies to Lewy Bodies (LB), which characterize neurodegeneration in Parkinson’s disease (PD) and Lewy Body Dementia (LBD). It is now established that alpha-syn-related cytopathology and inclusions do occur within catecholamine cells also following methamphetamine (METH) exposure (Vincent and Shukla
2023) in rodents (Fornai et al.
2004) and humans (Quan et al.
2005; Wu et al.
2021). This neuropathology observed in chronic METH abusers resembles to what observed in PD patients (Vincent and Shukla
2023). In detail, METH-induced neurodegeneration involves aggregation of alpha-syn proto-fibrils within catecholamine neurons and drives these neurons to make them more vulnerable to degeneration as recognized in Parkinson's disease (Vincent and Shukla
2023). In fact, wide cytosolic areas during parkinsonism and following METH exposure similarly stain for alpha-syn, poly-ubiquitin, and other antigens related to the autophagolysosomal pathways (Fornai et al.
2004; Forno
1996; Quan et al.
2005; Shimura et al.
1999; Spillantini et al.
1997; Wulf et al.
2022). This extends to cell-to-cell propagation of alpha-syn following METH, and deleterious effects of alpha-syn expression in METH-induced neurodegeneration (Ding et al.
2020; Meng et al.
2020; Wu et al.
2021). This is in line with the involvement of autophagy and autophagolysosomes in the damage of catecholamine cells (Anglade et al.
1997; Chandra et al.
2004; Ferrucci et al.
2008; Fornai et al.
2003,
2005; Larsen et al.
2002; Sato et al.
2018). An intense debate is ongoing concerning the intimate structure of alpha-syn-related cytopathology. In fact, the nature of neuronal inclusions is seminal to comprehend the neurobiology of disease and to dissect, which specific step needs to be targeted to plan disease-modifying therapies [either in neurodegeneration or in the addicted brain (Iacovelli et al.
2006; Mahul-Mellier et al.
2020)]. The task of deciphering the structure of neuronal inclusions and molecular components building up cytopathology within catecholamine cells would require a quantitative analysis of specific components. Unfortunately, a quantitative analysis in situ is quite complex and it was never carried out. Thus, inclusions are identified only by qualitative approaches, based on immuno-histochemistry or plain electron microscopy. To our knowledge, no quantitative assessment comparing specific proteins or non-protein components was carried out in situ. Such a measurement requires to be established in situ to avoid the bias of measuring dispersed cytosolic areas or nuclear components. Such an in situ investigation can be obtained solely by counts of immuno-gold stained antigens, which allow the stoichiometric detection of each specific component within its original cell compartment (Bergersen et al.
2008). So far, intense immuno-staining at light microscopy led to assume that alpha-syn represents the major component of the inclusions and the wide cytopathology developing widespread within degenerating catecholamine cells.
However, doubts remain concerning the specific protein amount just based on qualitative immuno-staining. This also leaves open a key question: how much alpha-syn compared with some other key protein does occur within inclusions? Similarly, the abundance of protein compared with non-protein components remains elusive. For instance, in the case of LB in PD, the recent work by Shahmoradian et al. (
2019) detailed the ultrastructure of specific areas selected at light microscopy as strongly stained for alpha-syn. Within these regions, a remarkable amount of non-protein structures covering a great area was identified at TEM. Similarly, pioneer ultrastructural studies about inclusions led to stain some key proteins (Iwatsubo et al.
1996) and spoke up for the occurrence of multi-faceted tubulo-vesicular vacuoles. However, in this pioneer work, immuno-gold quantification was not carried out and inclusions were not analyzed in situ, but they were inferred following a gradient centrifugation. Thus, at present days, even in keeping with the mainstream assessing alpha-syn as the major component of inclusions within diseased catecholamine neurons, the amount of this protein was never counted. Similarly, a quantitative comparison between alpha-syn and other key proteins remains non-investigated. Again, the amount of non-protein structures being abundant in these areas remains non-defined. The analysis of proteins other than alpha-syn is emerging in recent studies showing the potential key role of p62 and poly-ubiquitin in seeding neuronal inclusions at early stages of catecholamine cell pathology and persisting later on in the disease course (Kurosawa et al.
2016; Noda et al.
2022; Sato et al.
2018,
2020,
2021). Still, the lack of quantitative data concerning the amount of these specific proteins and the area covered by vesicular bodies distinct from protein aggregates is in need to be investigated. Therefore, in the present study, we counted stoichiometrically specific key proteins using immuno-gold transmission electron microscopy (TEM) within alpha-syn-rich pathological compartments. These areas correspond to those identified following immuno-staining at light microscopy in METH-treated catecholamine cells. We detailed the quantitative relevance of alpha-syn compared with some key proteins such as p62 and poly-ubiquitin and we measured the protein vs. non-protein components within these areas occurring during METH-induced cytopathology.
Materials and methods
Experimental design
Cells were administered various doses of METH to select the optimal dose to produce the most severe alpha-syn-related pathology, while avoiding to induce extensive cell death. At this purpose, a dose–response curve showing the occurrence of cell death was compared with a dose–response curve measuring alpha-syn-related cytopathology. The chance to analyze a highly reproducible in vitro system allows to count specific protein amount within alpha-syn positive areas. The ultrastructural analysis of these alpha-syn positive cell domains following METH was developed by combining light and electron microscopy (CLEM), similarly to that recently reported by Shahmoradian et al. (
2019). A preliminary approach consisted of selecting specific areas visualized at light microscopy through semi-thin sections to further proceed with electron microscopy to carry out a quantitative assessment. Semi-thin slices stained with toluidine blue provided a further analytical step to confirm the placement of those pale cytosolic areas observed at Hematoxylin & Eosin (H&E) light microscopy, which characterize METH-induced cytopathology, and corresponding to strong alpha-syn staining as confirmed by pre-embedding immuno-peroxidase. Once these areas were identified through semi-thin sections, they were further dissected to carry on ultra-thin slices for electron microscopy, where the amounts of specific antigens could be detected by immuno-gold stoichiometry. These pale eosinophilic areas represented by H&E and toluidine blue histochemistry were evidenced as encircled red-dotted line. Within these areas, which correspond to those previously immuno-stained at light microscopy, a further validation of alpha-syn content was provided by ultra-thin sections (70–90 nm) from samples undergone pre-embedding immuno-peroxidase before proceeding with the counts of immuno-gold stoichiometry of specific protein content and plain ultrastructural morphometry of membranous organelles.
Thus, the first part of the study assessed specific (alpha-syn, p62, poly-ubiquitin, ubiquitin) protein amount at light microscopy (immuno-peroxidase and immuno-fluorescence) following METH administration. The second part of the study combined light and electron microscopy (CLEM) to count protein stoichiometry at TEM within corresponding cytosolic areas possessing strong alpha-syn immuno-staining at light microscopy. In these cytosolic areas featuring a cytopathology reminiscent of inclusions, the amount of alpha-syn was compared with other proteins, such as p62 and poly-ubiquitin in situ. In fact, these latter proteins were recently claimed as early markers of catecholamine cytopathology (Kurosawa et al.
2016; Noda et al.
2022; Sato et al.
2018,
2020,
2021). This ultrastructural analysis indicates that, within alpha-syn abundant areas, p62 prevails instead. Therefore, we added a reversed sampling focusing also on those areas which expressed the highest content of p62. In both cases, areas were measured to calculate the membranous organelles (lysosomes, autophagosomes, and mitochondria) compared with protein content.
Cell cultures
Pheochromocytoma PC12 cell cultures, purchased from IRCCS San Martino Institute (Genova, Italy), were kept in a wet atmosphere with 5% CO
2 at 37 °C and were grown in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with horse serum (HS, Sigma-Aldrich), fetal bovine serum (FBS, Sigma-Aldrich), and antibiotics (streptomycin and penicillin). Experiments were carried out when PC12 cells were in the log-phase of growth, corresponding to 70% confluence (Qiao et al.
2001; Song et al.
1998). Before experimental treatments, cells were seeded according to the different experimental procedures and incubated for 24 h at 37° C in 5% CO
2. In detail, for trypan blue (TB) staining, cells were seeded at a density of 10
4 cells/well and placed within 24-well plates in 1 mL of culture medium; for light microscopy staining procedures, 5 × 10
4 PC12 cells were seeded on poly-lysine coverslips and placed in 24-well plates in a final volume of 1 mL/well. For TEM, 1 × 10
6 cells were seeded in culture dishes in a final volume of 5 mL.
Cell treatments
A stock solution of METH (kindly gifted by Forensic Medicine, University of Pisa) 10 mM was obtained by dissolving in 1 mL of culture medium 2.3 mg of METH. Aliquots of the stock solution were diluted in the culture medium to obtain the treatment solutions. In detail, PC12 cells were exposed to increasing doses of METH, ranging from 1 up to 1000 µM, for 72 h. Control cultures were kept in the same volume of culture medium for the same time interval. This time interval was selected based on previous studies (Fornai et al.
2004; Lazzeri et al.
2018,
2021). At the end of the treatments, PC12 cells were washed in PBS and processed according to the various experimental procedures. After the pilot dose–response study, the dose of METH which was selected to analyze various antigens and ultrastructural morphometry was 100 µM for 72 h.
Hematoxylin and eosin (H&E) histochemistry
Cells were fixed in a 4% paraformaldehyde phosphate-buffered solution (PBS) for 15 min, washed in PBS, and then immersed for 15 min in the hematoxylin solution (Sigma-Aldrich). The hematoxylin staining was stopped through repeated washing in running water. After, cells were immersed within the eosin solution (Sigma-Aldrich) for a few minutes and washed out again to remove the excess of dye. Finally, after dehydration in increasing alcohol solutions, cells were clarified in xylene, covered with DPX mounting medium (Sigma-Aldrich) and observed under a Nikon Eclipse 80i light microscope (Nikon, Tokyo, Japan) equipped with a digital camera connected to the NIS Elements software for image analysis (Nikon, Tokyo, Japan).
Cell count was carried out under light microscope, using a 20 × magnification; the number of H&E-stained cells in each experimental group was counted and expressed as the mean percentage ± SEM of the control group (which corresponds to 100%). Data refer to three independent experiments.
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Fluoro-Jade B (FJB) histo-fluorescence
Cells were fixed with a solution of paraformaldehyde 4% for 5 min, washed in PBS, and immediately incubated with 0.06% potassium permanganate for 10 min at room temperature. After washing in distilled water, cells were incubated for 20 min in a FJB solution prepared by dissolving 0.01% FJB (Merck Millipore, Billerica, MA, USA) in acetic acid. Cells were incubated with 0.0004% of this FJB solution for 20 min and then cover-slipped with mounting medium. FJB-positive cells were analyzed using a Nikon Eclipse 80i light microscope (Nikon, Tokyo, Japan), equipped with a florescence lamp and a digital camera connected to the NIS Elements software for image analysis (Nikon, Tokyo, Japan).
Cell count was carried out under fluorescence microscope at 20 × magnification. The number of FJB-fluorescent cells was expressed as the mean number ± SEM for each experimental group. The intensity of the fluorescent signal was measured under florescence microscopy using the software Image J (NIH, Version 1.8.0_172, Bethesda, MD, USA) and values are expressed as the mean percentage ± SEM of optical density (assuming controls as 100%) from N = 90 cells/group. Data refer to three independent experiments.
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Trypan blue (TB)
Cells were collected and centrifuged at 800×g for 5 min, and the cell pellet was suspended in 0.5 mL of culture medium. Twenty-five µL of cell suspension was incubated for 10 min in a solution containing 1% TB in PBS and 10 µL of this solution were injected into a Bürker chamber and analyzed under an Olympus CKX 41 inverted microscope (Olympus Corporation, Tokyo, Japan). Viable and nonviable cells were counted and values were expressed as the mean percentage ± SEM of TB-positive cells out of the total cells. Data refer to three independent experiments.
Immuno-histochemistry
After washing in PBS, PC12 cells were fixed with 4% paraformaldehyde in PBS for 15 min, and incubated with 0.1% TritonX-100 (Sigma-Aldrich) for 15 min in PBS.
For immuno-fluorescence experiments, cells were immersed for 1 h in a blocking solution containing 10% normal goat serum (NGS) in PBS at room temperature and then were incubated overnight at 4 °C in a solution containing the primary antibodies in PBS and 1% normal goat serum. In detail, the following primary antibodies (AbI) were used: (1) anti alpha-syn AbI (Abcam, Cambridge, UK), diluted 1:100; (2) anti-p62 AbI (Abcam), diluted 1:100; (3) anti-poly-ubiquitin AbI (Abcam), diluted 1:100; (4) anti-ubiquitin AbI (Sigma-Aldrich), diluted 1:100.
After rinsing in PBS, cells were incubated for 1 h with the appropriate fluorophore-conjugated secondary antibodies (i.e., Alexa 488, Life Technologies Carlsabad, CA, USA, or Alexa 594, Life Technologies) diluted 1:200. All these reactions were carried out within the well plate. After washing in PBS, cells were transferred on coverslip, mounted with the mounting medium Fluoroshield (Sigma-Aldrich), and finally observed under the Nikon Eclipse 80i light microscope (Nikon) equipped with a fluorescent lamp and a digital camera connected to the NIS Elements Software for image analysis (Nikon). Negative control cells were incubated with secondary antibodies only. For double fluorescence pictures, single fluorescent images were acquired independently, and then, they were merged using the NIS Elements Software (Nikon).
For immuno-peroxidase experiments, cells were incubated in 3% hydrogen peroxide (H2O2) for 20 min at room temperature to block endogenous peroxidase activity, and then were plunged in a blocking solution containing 10% NGS in PBS for 1 h at room temperature. Cells were incubated overnight at 4 °C with the primary antibody solution containing 2% NGS in PBS and the following AbI: the anti-alpha-syn AbI (Abcam) (1:2000), the anti-p62 AbI (Abcam, 1:2000), the anti-poly-ubiquitin AbI (Abcam, 1:2000), and the anti-ubiquitin AbI (Sigma-Aldrich, 1:2000).
The antigen–antibody reaction was revealed using the appropriate biotin-conjugated secondary antibodies (Vector Laboratories, Burlingame, CA, USA) diluted 1:200 for 1 h at room temperature, followed by avidin–biotin complex (Vector) for 1 h and the peroxidase substrate diaminobenzidine (DAB, Vector) for a few minutes. Finally, cells were dehydrated using increasing alcohol solutions. All these reactions were carried out within the well plate. After washing in PBS, cells were clarified in xylene and transferred on coverslips where DPX mounting medium (Sigma-Aldrich) was added before their observations at light microscopy (Nikon) equipped with a digital camera connected to the NIS Elements software for image analysis (Nikon, Tokyo, Japan). Negative control cells were incubated with secondary antibodies only.
The optical density of each single fluorescent or peroxidase-stained picture was measured using Image J software (NIH, Version 1.8.0_172, Bethesda, MD, USA). Values are given as the mean percentage ± SEM from N = 90 cells/group.
In double fluorescent pictures, merging areas were measured in µm2 using Image J software (NIH, Version 1.8.0_172, Bethesda, MD, USA) and values are given as the mean merging areas ± SEM per cell from N = 90 cells/group.
All data refer to three independent experiments.
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Sample preparation for semi-thin slices and combined light and electron microscopy (CLEM)
At the end of the treatment, cells were centrifuged at 1000×g for 5 min, rinsed in PBS, and fixed in a solution of 2.0% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M PBS (pH 7.4) for 90 min at 4 °C. Cells were then washed out in PBS (0.1 M), post-fixed in 1% osmium tetroxide (OsO4) for 1 h, at 4 °C, and dehydrated in increasing ethanol solutions. Finally, they were embedded in Epoxy resin.
Semi-thin slices (about 1 µm thick) were cut by ultra-microtome (Leica Microsystems, Leica Microsystems, Wetzlar, Germany), and they were stained with toluidine blue and observed at light microscopy (Nikon). These slices were ordered with ultra-thin slices (70–90 nm thick) to combine light and electron microscopy (CLEM) analysis of the corresponding areas. Ultra-thin slices (70–90 nm thick) were counterstained with uranyl acetate and lead citrate to be examined using a JEOL JEM SX100 transmission electron microscope (JEOL, Tokyo, Japan).
To provide internal validation, each semi-thin slice was followed by an ultra-thin slice (each including five series), which were processed for light and electron microscopy, respectively. This allowed to compare similar cytosolic areas according to a roughly 0.1 µm thickness interval between light and electron microscopy sampling. As a reference point, in these alternate slices, we used as a spatial reference the shape of vacuolated cytosolic domains, which were poorly stained by toluidine blue (light beam) and provided a poor contrast to the electron beam. The choice of selecting highly vacuolated cytosolic domains highly stained with alpha-syn was based on light microscopy data. In fact, the occurrence of clusters of alpha-syn was abundant in these cell regions. Immuno-peroxidase carried out post hoc at pre-embedding validated the occurrence of these antigens within ultra-thin slices.
Pre-embedding for immuno-peroxidase
At TEM, alpha-syn and p62 were also labeled by immuno-peroxidase. Cell pellets were fixed with 2.5% paraformaldehyde and 0.1 glutaraldehyde dissolved in PBS for 90 min. After washing in PBS, cell pellets were incubated in 0.002% hydrogen peroxide in 0.05 M Tris–HCl buffer, pH 7.6 for 2.5 min. Then, after washing in PBS, cell pellets were permeabilized in ethanol (10% for 5 min, 25% for 5 min, and 10% for 5 min) and pre-blocked with a solution containing 10% NGS and 0.2% saponin in PBS for 30 min. Samples were then incubated with anti-alpha-syn AbI (1:100, Abcam) or anti-p62 AbI (1:100, Abcam) diluted in 10% NGS and 0.2% saponin in PBS for 24 h. Then, samples were incubated with a solution containing the biotin-conjugated secondary antibodies (Vector) diluted 1:100 for 1 h at room temperature. After washing in PBS, samples were incubated in avidin–biotin peroxidase complex (Vector) for 1 h. After washing in PBS samples were incubated in 0.075% DAB (Vector) for a few minutes.
Samples were washed in PBS and osmicated, dehydrated, and embedded in Epoxin resin. Ultra-thin sections were cut by ultra-microtome (Leica Microsystems) and were observed under TEM (JEOL JEM SX100, JEOL, Tokyo, Japan).
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Post-embedding for immuno-gold TEM
Ultra-thin slices from cell cultures were collected on nickel grids, and they were de-osmicated in aqueous solution saturated by sodium metaperiodate (NaIO4), for 15 min. Sections were washed three times for 10 min in ice-cold filtered PBS (pH 7.4) and the grids were treated with ice-cold PBS containing 10% NGS and 0.2% saponin to block non-specific antigens for 20 min at room temperature.
Primary antibodies were incubated in ice-cold PBS containing 1% NGS and 0.2% saponin in a humidified chamber overnight, at 4 °C. The following primary antibodies were used: anti-alpha-syn AbI (1:100, Abcam); anti-p62 AbI (1:100, Abcam); anti-poly-ubiquitin AbI (1:100, Abcam); anti-ubiquitin AbI (1:100, Sigma-Aldrich). Double immuno-gold staining was used to compare the amount and co-localization of the following: alpha-syn and p62, alpha-syn and poly-ubiquitin, and p62 and poly-ubiquitin.
Stoichiometry staining was obtained through a solution containing gold-conjugated secondary antibodies (gold particle diameter, 10 nm or 20 nm, BB International, Cardiff UK) diluted 1:100, in PBS containing 1% goat serum and 0.2% saponin for 1 h, at room temperature. The size of immuno-gold particles was switched to validate the non-relevance of steric encumbrance in double immuno-staining. After rinsing in PBS, grids were incubated in 1% glutaraldehyde for 3 min, and they were washed in distilled water and further stained with uranyl acetate and lead citrate. Ultra-thin sections were finally observed at TEM (JEOL JEM SX100). Control sections were incubated with secondary antibodies only.
The number of immuno-gold particles related to alpha-syn, p62 and poly-ubiquitin proteins was expressed as the mean ± SEM within 2 µm2 selected areas from n = 30 cells per group.
Area of the tubulo-vesicular membranes and the cytosol within 2 µm2 selected areas was measured using Image J software (NIH, Version 1.8.0_172, Bethesda, MD, USA) at 6000 ×. Values are given as the mean percentage ± SEM from n = 30 cells/group.
Electron density of 2 µm2 selected areas was measured using Image J software (NIH, Version 1.8.0_172, Bethesda, MD, USA) at 6000 ×. Values are given as the mean percentage ± SEM of electron density measured in METH-treated cells compared with electron density measured in control cells (assumed as 100%) from n = 30 cells/group.
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Extended statistics
In this paragraph, we report classic descriptive and inferential statistics, which were implemented by an extended explanation of arbitrary criteria used here to provide the optimal sampling for each procedure. A brief comment about validation of the procedure is provided as well. The effects of various doses of METH ranging from 1 to 1000 µM on cell damage (H&E) were expressed as the mean percentage of healthy cells ± SEM compared with control. The number of degenerating cells (FJB) was expressed as the mean ± SEM of stained cells. Degenerating cells visualized at TB was expressed as the percentage of stained cells within the whole population. The difference between groups were assessed by ANOVA with Sheffe’s post hoc analysis; H0 was rejected for p < 0.05.
The issue of combining different procedures and different techniques to count cell damage at light microscopy may produce discrepancies based on the specific procedure. For instance, the amount of severe cell damage assessed by H&E is based on the actual lack of cell structures (unique among light microscopy procedures used here) and the presence of remarkable alterations of cell shape and size and faint cytosol visible as pale eosinophilic cytosolic areas. In keeping with H&E staining, the amount of cell damage included those cells, where these alterations were severe. In the case of FJB, the count is based on the fluorescent area conventionally assuming that FJB immuno-fluorescence is synonymous of dying cell (which still may not be constantly true considering our lack of an in-depth knowledge of which and how many markers are responsible for FJB-induced fluorescence). In this case, additional bias may lead to non-damage dependent occurrence of some molecules, which target FJB, thereby providing a bias. This may explain why the amount of cell death assessed using FJB was the highest compared with all other methods (still considering that this may also depend on the highest sensitivity of this procedure). Data about cell death counted at TEM were inferred by counting the decrease of viable cells compared with control. Despite slight differences, the consistency across different techniques and between light and electron microscopy was remarkable concerning the amount of METH-induced cell damage, and values were quite steady, which internally validate each single procedure applied here.
Data about immuno-fluorescence for alpha-syn, p62, poly-ubiquitin, and ubiquitin stained alone or in combination (merging) were expressed as semi-quantitative fluorescent densitometry considering the fluorescent area of merging comparing controls with METH 100 µm. The comparison was carried out using ANOVA with Sheffe’s post hoc analysis;
H0 was rejected for
p < 0.05. This rough calculation due to intrinsic limits of non-linear relationship between protein amount and immuno-staining serves as a guide to better address molecular quantification of these proteins at TEM. Alpha-syn positive areas were selected by counting immuno-gold particles within selected circular areas of 2 µm
2. This size was selected based on sampling the distribution of immuno-gold clusters observed in alpha-syn hot spots at CLEM in METH-treated cells. These consistently overlap with pale eosinophilic areas. The amount of immuno-gold for alpha-syn within these areas was different within various cytosolic domain ranging from 0 up to 12 immuno-gold. An arbitrary cut-off was set at 7 immuno-gold alpha-syn particles which was arbitrarily selected to define at sub-cellular level a cluster of alpha-syn. Within these areas of high alpha-syn content, the amount of p62 was counted as well. In this case, we used different immuno-gold particles owing different diameter (either 10 nM or 20 nM, respectively) to distinguish both antigens in the same ultra-thin section. In parallel experiments, we assessed that the diameter of the immuno-gold particles did not affect the count of the antigen, likely due to a lack of allosteric interference between the immuno-gold particles and primary antibodies on antigen epitopes. The comparison between p62 and alpha-syn immuno-gold was carried out using ANOVA with Sheffè’s post hoc analysis. Since p62 prevails at large compared with alpha-syn even in those areas being selected as the richest in alpha-syn content, a second step was necessary to re-assess the relative amount of these proteins by starting to select areas where p62 was most abundant (cut-off 100 immuno-gold particles per 2 µm
2), independently by the amount of alpha-syn. Despite some overlapping between these antigens occurs, we found that a different placement was evident at electron microscopy, which did not emerge when the localization of both antigens was detected at light microscopy. In fact, the richest p62 immuno-gold areas analyzed at TEM may not contain high alpha-syn. Again, at TEM compared with light microscopy, the occurrence of p62 was strikingly more abundant than alpha-syn. Such a difference was magnified when alpha-syn and p62 richest areas were calculated following METH administration. In these experimental conditions, these 2 µm
2-wide areas were analyzed concerning their structure. In fact, within these areas, the amount of altered lipid membranous organelles was counted as well. In detail, the area taken by membrane limited organelles (autophagosomes, lysosomes and mitochondria) was calculated independently within alpha-syn and p62 rich areas. Despite some areas feature a high content of both antigens, we found that vesicles limited organelles were more abundant within areas identified by p62 immuno-gold staining compared with alpha-syn immuno-gold staining. Within these areas, the mean electron-density was calculated. In calculating the specific kind of membranous organelles, we purposefully did not discriminate between the amounts of mitochondria compared with lysosomes or autophagosomes to harvest all membranous structures as previously described (Shahmoradian et al.
2019). The nuclear area was never considered in keeping with the cytosolic nature of the pathological process under analysis. In selecting the placement, the counted area was placed around the protein cluster under primary analysis. This led to discrepant, only partially overlapping regions depending on which protein was primarily counted. Again, a discrepancy exists considering non-protein vesicle crowding. This partial overlapping indicates that the region shape and size varies depending on which structure is considered as the hallmark. The choice of the protein hallmark was done at first based on classic literature (alpha-syn), and then, it was slightly modified based on actual quantitative findings (the excess of p62). Concerning the pattern of p62, we noticed a remarkable packing where p62 and poly-ubiquitin were expressed densely. The size of these small circular areas providing a sort of p62/poly-ubiquitin domain was roughly tenfold lower compared with the circles selected for alpha-syn clusters. Therefore, a further count was used within these 200 nm
2 areas to better express the density of p62 and to make a comparison with the density of alpha-syn.
From a statistical perspective and a methodological approach, we found strong discrepancies between immuno-fluorescence/immuno-peroxidase at light microscopy and immuno-gold at TEM. In detail, at light microscopy, most cells appear to stain for alpha-syn and p62 according to an all (METH) or none (control) pattern. This contrasts with TEM showing a twofold difference between controls and METH. This is true also concerning various cell domains. This suggests that the statistical power of TEM to paint a scenario which is adherent to actual cytopathology is enormous compared with light microscopy. This latter procedure appears to provide negligible staining up to a level where a sort of dramatic staining enhancement takes place.
The striking discrepancy between immuno-stained area observed at light microscopy (almost as a clear-cut region compared with surrounding cytosol) with the faint, undefined border under TEM is due to the striking difference in magnification, which either neglects or highlights the continuum of cytopathology leading to undefined borders for these multi-faceted biological structures observed at high magnification.
Discussion
The present manuscript provides a specific analysis about some key features of cell pathology produced by METH within catecholamine neurons resembling cytopathology, which develops within catecholamine neurons in PD. The detailed analysis of catecholamine cell pathology including inclusions represents a crucial step to understand the neurobiology of diseases such as neurodegenerative disorders affecting catecholamine neurons and the pathology of the addicted brain. A few studies were aimed to characterize these features until the end of the past century. A pioneer study was carried out by Hirsch et al. (
1985). The study was designed to purify an antibody specific for LB in PD. Such an approach was followed by the works by Pollanen et al. (
1992,
1993,
1994) and the isolation of poly-ubiquitin rather than ubiquitin within LB by Iwatsubo et al. (
1996). Soon after, the occurrence of alpha-syn was reported by Spillantini et al. (
1997) and such a protein was considered as the major component of LB. The occurrence of alpha-syn is impressive when light microscopy immuno-detection is carried out or when samples are pre-treated with protease and TEM is carried out on protease resistant tissue centrifugates. Nonetheless, at present, no study compared the stoichiometric amount of alpha-syn with other proteins during cytopathology of catecholamine neurons in situ. Therefore, conclusions were largely based on qualitative data. In keeping with such a qualitative analysis, novel approaches using CLEM led to identify an impressive amount of non-protein aggregates within diseased catecholamine neurons, where a number of membranes and vesicular organelles seem to be densely represented (Shahmoradian et al.
2019). Still even in this case, the qualitative approach prevails over data quantification. Therefore, the present study specifically investigated the stoichiometric amount of a few critical proteins including alpha-syn by profiting of an in vitro system administered METH.
Data provided by light microscopy indicate that alpha-syn accumulates dose-dependently following 72 h of METH exposure. This is consistent with data we previously reported in vivo and in vitro, where METH was shown to induce alpha-syn positive inclusions in catecholamine cells of substantia nigra and catecholamine cell lines (Fornai et al.
2004). Remarkably, as reported here, other key antigens involved in the degeneration of catecholamine-containing neurons such as p62 and poly-ubiquitin increase according to a roughly similar dose–response curve. In keeping with cell pathology of catecholamine neurons, the occurrence of poly-ubiquitin appears to be way more relevant than ubiquitin alone as reported by pioneer works (Iwatsubo et al.
1996). In the present manuscript, evidence is provided that dose-dependent increase in alpha-syn immuno-fluorescence is shifted to the left compared with dose-dependent METH-induced cell death. The latter was assessed by three independent methods (H&E, TB, FJB) which given similar results, thus providing an internal validation across various techniques. In particular, the strong effects obtained following FJB histochemistry indicate such a marker as a reliable tool to assess cell degeneration even in vitro following METH. Remarkably, cell damage assessed by FJB histochemistry is slightly more dramatic compared with H&E and TB. As briefly mentioned in the results section, this may also depend on the specific targets of FJB, which may stain molecules other than those strictly involved in cell degeneration and still induced by METH exposure such as specific poly-amines.
Immuno-fluorescence and immuno-peroxidase carried out following a dose of METH selected to increase key proteins in the absence of frank cell death indicate a marked increase of alpha-syn. The METH-induced increase in alpha-syn is concomitant with an increase of p62 and poly-ubiquitin. When roughly assessed at densitometry (both immuno-peroxidase and immuno-fluorescence), the increase of p62 and poly-ubiquitin in METH-treated cells is roughly twofold compared with the increase in alpha-syn.
In the second part of the study, light microscopy was matched with ultrastructural stoichiometry through an experimental approach combining light and electron microscopy (CLEM) as recently applied, to provide a qualitative analysis of the intimate structure of alpha-syn-positive LB in catecholamine neurons by Shahmoradian et al. (
2019). Such an approach was used here by starting from the identification of pale eosinophilic areas identified at H&E, to focus on specific regions where semi-thin toluidine blue-stained sections confirm that pale cytosol areas were stained by immuno-peroxidase for alpha-syn or p62. Within these selected cell spots, immuno-gold and plain electron microscopy allowed to dissect by stoichiometry quantitative amount of specific proteins occurring following METH administration. Similarly, the fine ultrastructure of cell regions possessing a high amount of these proteins was assessed. In keeping with recent data about cell pathology of catecholamine neurons such as LB from PD patients, here, we found an impressive amount of membranous vesicles within critical spots either staining for alpha-syn and/or p62. It was remarkable that these cell regions possess an amount of p62 molecules within 2 µm
2 areas surpassing at large (tenfolds) the amount of alpha-syn counted within areas of the same size. Again, the increase in p62 was concomitant with poly-ubiquitin. In fact, these antigens where densely packed providing a cell domain, which indeed was clustered within smaller regions (200 nm
2) compared with those featuring increased alpha-syn immuno-staining. This indicates that the amounts of p62/poly-ubiquitin molecules within specific clusters reach a density of roughly 100-fold compared with the density of alpha-syn. The occurrence of high amounts of p62/poly-ubiquitin way in excess compared with alpha-syn was quite impressive when comparing immuno-gold stoichiometry (100-fold) with rough immuno-fluorescence densitometry (twofold). The size of these p62/poly-ubiquitin-rich clusters was much smaller compared with the scattered clusters of alpha-syn. Again, the relationship of p62/poly-ubiquitin with abnormal amounts of membranous organelles surpasses the association between organelles and alpha-syn. These data indicate the quantitative composition of specific protein and non-protein content within cell domains during METH-induced pathology. The present data also emphasize the different information between semi-quantitative densitometry roughly esteemed at light microscopy and the stoichiometry quantification of authentic protein amounts and tubulo-vesicular areas detected at TEM. This strong discrepancy explains technical and conceptual uncertainties in the definition of catecholamine cell pathology. This is likely to rely on the non-linear relationship between fluorescence/peroxidase signal and protein amount compared with specificity of immuno-gold stoichiometry. A limit of the present data, which needs to be taken into account concerns the administration of METH within catecholamine cell cultures. Nonetheless, there is a remarkable consistency with data obtained ex vivo within nigral catecholamine neurons of transgenic mice expressing PD-inducing genes, which were recently reported to sort similar results. In fact, the occurrence of p62 and poly-ubiquitin appears within LB-like bodies at early time intervals (2 months), while alpha-syn joins the inclusions only at 9 months of age. These led the authors to suggest that the seeding of pathological inclusions may be represented by p62 rather than alpha-syn (Kurosawa et al.
2016; Noda et al.
2022; Sato et al.
2018,
2020,
2021). The occurrence of abundant lipid-containing membranous structure, focally, close to the p62 and poly-ubiquitin clusters and to alpha-syn molecules matches the qualitative data recently obtained by Shahmoradian et al. (
2019) concerning the cell pathology of nigral neurons from PD patients, where the composition of LB was extremely variable and rich in lipid membranes and various organelles.
This is quite remarkable and indeed unexpected considering that alpha-syn is indicated as the major component of a number of neuronal inclusions. Such a discrepancy may depend on the use of METH or the specific cell line. Again, technical differences between light and electron microscopy may further explain a number of issues which affect the semi-quantitative measurement of immuno-staining compared with stoichiometry measurement at TEM. All these issues may contribute to decipher why, despite the amount of alpha-syn stoichiometrically measured is 100-fold less dense, the fluorescence or peroxidase is very intense; for instance: (1) the close space between p62 and poly-ubiquitin proteins does not allow an easy access to peroxidase- or fluorophores-conjugated antibodies; (2) the binding sites of p62 and poly-ubiquitin are overwhelmed by an excess of endogenous protein substrates competing with primary and secondary antibodies; (3) the co-chaperonine nature of alpha-syn may provide multiple binding sites, which in turn may attract a high number of primary antibodies; (4) the baseline recruitment and buffering of p62 and poly-ubiquitin is elevated even in baseline conditions due to a powerful engagement of both these molecules; (5) the recruitment of alpha-syn in baseline conditions by endogenous proteins is much less intense; (6) in addition, the density of alpha-syn may produce an optimal distance to detect a strong signal-to-noise ratio for the fluorophore/peroxidase bound to secondary antibodies. All these issues potentially altering the authentic protein amount are erased by TEM stoichiometry. In fact, a main focus of the present study is to provide the authentic protein amount by in situ protein measurement (Bergersen et al.
2008). Still, even TEM detection may introduce other bias such as different access to antigens of immuno-gold particles depending on their size due to mutual steric encumbrance, where larger immuno-gold particles are underscored compared with smaller particles, which may lead either to mask or unmask double staining. Such an issue was approached by reverting the size of immuno-gold particles binding the primary antibodies. When the size of immuno-gold used to stain two antigens was switched, the data were confirmed. In fact, the authentic amount of p62, and alpha-syn did not vary when the size of immuno-gold particles was reverted. One major outcome of the study is the demonstration that p62/poly-ubiquitin, apart from being more abundant in baseline conditions compared with alpha-syn, undergo an upregulation and clusterization following METH, which surpasses at large the increase, which is measured for alpha-syn. This clusterization occurs preferentially as densely packed clusters within electron-rare cytosolic regions, where the ultrastructure reveals a high tubulo-vesicular component mainly made up of autophagosomes/omegasomes and mitochondria. This is in line with the physiological role of p62, which acts as a shuttle to import protein and other substrates including mitochondria through a poly-ubiquitin chain within the nascent autophagosome (Cohen-Kaplan et al.
2016). Thus, the accumulation of p62/poly-ubiquitin is supposed to occur where an impairment of the autophago-lysosome system takes place. This is in line with the molecular effects of METH (Lazzeri et al.
2018) and it is strikingly similar to the impairment of these organelles as emerging in recent studies in PD patients. This explanation inherently addresses the second main finding of the present work which revels the paucity of protein compared with membranous organelles to build up the fine neuropathology induced by METH within catecholamine neurons. Since this occurs also in PD brain, it is not surprising that the mimicking of parkinsonian neuropathology produced by METH reproduces quite similar findings. Of course, from a methodological stand point, we are obliged to leave the benefit of the doubt concerning a profound diversity between LB and METH-induced inclusions, although some literature tend to overlap these conditions (Kousik et al.
2014). In any case, this fully applies to the pathology of catecholamine neurons in the addicted brain, where a long-term METH abuse predisposes to develop PD (Kousik et al.
2014; Lappin and Darke
2021; Rumpf et al.
2017; Shin et al.
2021; Vincent and Shukla
2023).
The advantage of the present study relies on applying CLEM in vitro where the high reproducibility and flexibility of the model allows to count single protein molecules. In fact, previously, CLEM was used ex vivo to analyze qualitatively the structure of LB without assessing the amount of specific proteins by immuno-gold. In these conditions, it is difficult to preserve antigen conformation within human post-mortem tissue. However, in 1996, immuno-gold was used to measure ubiquitin in post-mortem human brain tissue. Moreover, the immuno-gold was not used to quantify the antigen and it was not carried out in situ within specific cell domain, since it was used in cell fractions following centrifugation in sucrose gradient (Iwatsubo et al.
1996). Again, this study was carried out before alpha-syn was recognized as a marker of neuropathology, and at that time, p62 was not explored in neurodegeneration. Therefore, this pioneer study remained seminal to emphasize the poly-ubiquitin rather than the ubiquitin staining of PD inclusions leaving unsolved the amount of p62 and alpha-syn. The present manuscript shed light on a primary role of p62 in METH toxicity, which is reciprocated by recent literature obtained from mice models expressing a number of genes inducing PD (Kurosawa et al.
2016; Noda et al.
2022; Sato et al.
2018,
2020,
2021). These data show that p62 and poly-ubiquitin appears within LB-like bodies at early time intervals (2 months) while alpha-syn joins the inclusions only at 9 months of age. This is fascinating in the light of recent findings obtained in a variety of experimental condition including PD brain, which show how a primary dysfunction of p62 leads to autophagy inhibition and subsequent alpha-syn accumulation and secretion and PD spreading (Oh et al.
2022).