Histological Examination of Mitochondrial Morphology in a Parkinson's Disease Model

Journal: Journal of Visualized Experiments 2023 Jun 23:(196). doi: 10.3791/65453. Title: Histological Examination of Mitochondrial Morphology in a Parkinson's Disease Model Authors: Dalila Ciceri, Lierni Gregorio-Zabala, Xabier Llama-Pino, Begüm Kurt, Jon OlanoBringas, Patricia Villegas-Zafra, Nora Bengoa-Vergniory PMID: 37427919 DOI: 10.3791/65453 TITLE: Histological examination of mitochondrial morphology in a Parkinson’s disease model AUTHORS AND AFFILIATIONS: Dalila Ciceri1*, Lierni Gregorio-Zabala1*, Xabier Llama-Pino1, Begüm Kurt1, Jon Olano-Bringas1, Patricia Villegas-Zafra1 and Nora Bengoa-Vergniory1,2,3,4 1 Achucarro Basque Center for Neuroscience, Leioa, Spain 2 University of the Basque Country (UPV/EHU), Department of Neuroscience, Leioa, Spain 3 Ikerbasque - Basque Foundation for Science, Bilbao, Spain 4 Oxford Parkinson’s Disease Centre and Department of Physiology, Anatomy and Genetics, University of Oxford, South Park Road, Oxford OX1 3QU, United Kingdom * Equal contribution SUMMARY: In this study we present a method to analyze the morphology of mitochondria based on immunostaining and image analysis in situ in mouse brain tissue. We also describe how this tool allow us to detect changes in mitochondrial morphology induced by protein aggregation in models of Parkinson’s Disease. ABSTRACT: Mitochondria play a central role in the energy metabolism of cells, and their function is especially important for neurons due to their high energetic demand. Therefore, mitochondrial dysfunction is a pathological hallmark of various neurological disorders, including Parkinson’s Disease. The shape and organization of the mitochondrial network is highly plastic which allows the cell to respond to environmental cues and needs; in addition, the structure of mitochondria is also tightly linked to their health. Here, we present a protocol to study mitochondrial morphology in situ, based on immunostaining of the mitochondrial protein VDAC1 and subsequent image analysis. This tool could be particularly useful for the study of neurodegenerative disorders, as it is able to detect subtle differences in mitochondrial counts and shape induced by aggregates of alpha-synuclein, an aggregation-prone protein heavily involved in the pathology of Parkinson’s Disease. This method has allowed us to report that substantia nigra pars compacta dopaminergic neurons harboring pS129 lesions have a reduced Aspect Ratio (AR) compared to their healthy neighboring neurons in a pre-formed fibril intra-cranial injection Parkinson’s model. INTRODUCTION: The central nervous system has an intense demand for ATP: neurons use ATP to support ionic gradients, neurotransmitter synthesis, synaptic vesicle mobilization, release, and recycling, and to enable local protein translation and degradation. More than 95% of ATP used by the brain is produced by the mitochondria (1). It is therefore not surprising that mitochondrial dysfunction is particularly harmful for neurons. In fact, mitochondrial function impairments play an important role in several neurological diseases, including neurodegenerative conditions, such as Parkinson’s Disease (PD) and Alzheimer’s Disease (AD) (2, 3). Multiple genes unequivocally linked to familiar PD encode proteins that are relevant for mitochondrial function and homeostasis, such as Parkin (4, 5, 6), PTEN-induced kinase 1 (PINK1) (7, 8) and DJ-1 (9). Further evidence for a role for mitochondrial dysfunction in PD is that treatments with inhibitors of the Complex I of the mitochondrial electron transport chain (Rotenone and MPTP) recapitulate several aspects of PD in vitro and in vivo (10). However, it is important to state that many pathological processes concur in provoking neuronal loss in PD, together with mitochondrial deficits: oxidative stress, altered calcium homeostasis, failure of the ubiquitin-proteasome and of autophagy-lysosomal systems and protein aggregation are amongst the most studied (reviewed in 11, 12 and 13). Mitochondria are heterogeneous in their shape: in addition to individual units, they are commonly found as extended reticular and tubular networks. The structure and the cellular location of mitochondria are critical for their function (14); in fact, mitochondrial networks are extremely dynamic, undergoing frequent processes of fission, fusion and mitophagy in order to serve the needs of the cells and to respond to environmental cues (15, 16). In addition, the morphology of mitochondria is intimately linked to their health status. For example, in human optic atrophy, genetic mutations that reduce mitochondrial activity lead to abnormal, slender and hyper-fused mitochondria (17). On the other hand, a variety of human diseases present aberrant mitochondrial morphology, including mitochondrial fragmentation or excessive mitochondrial fusion, which have deleterious effects on mitochondrial function (reviewed in 18). In the context of PD, we as well as others have previously shown that abnormal mitochondrial shape correlates with dysfunction in response to alpha-synuclein aggregates (19). While mitochondrial morphology has been extensively studied in vitro both in the context of PD and other diseases (20, 21, 22) protocols for the evaluation of mitochondrial morphology from in vivo sections, are lacking. This makes the study of mitochondria in vivo in the context of diseases such as PD highly dependent on transgenic animals (23) or the evaluation of midbrain extracts, which cannot provide cellular resolution. Here, we propose a protocol to study mitochondrial morphology in situ as an indicator of their functional status and health, based on immunostaining of the mitochondrial protein VDAC1 (24), followed by image analysis in paraffin-embedded tissue sections. We also show the results of this protocol in in vitro and in vivo well-characterized PD models: neuroblastoma cells overexpressing SNCA and brain tissue from mice subjected to intracranial injection of alpha-synuclein PreFormed Fibrils (PFFs). Co-immunostaining with an antibody against alpha-synuclein (in cells) or phosphoSer129-alpha-synuclein (in mouse brains) allowed us to identify the cells with protein aggregate pathology (overexpressed α-synuclein and α-synuclein fibrils, respectively) in our samples, while negative cells served as a non-pathological control within the same samples. Through this analysis we found that cells overexpressing SNCA or presenting pS129 lesions have a reduced aspect ratio, indicating fragmentation of mitochondria. PROTOCOL: All the procedures described in this section have been performed according to the Ethical framework provided by the University of the Basque Country Reference M20/2022/212, the Government of the Basque Country, the Spanish Government, and the European Union. Method 1: Mitochondrial morphology analysis by immunofluorescence of SNCA-overexpressing SH-SY5Y cells Here we briefly describe the generation of the studied in vitro material for the study, which will serve as a comparison for the in situ obtained results. It is recommended that this type of analysis is performed before launching an in vivo experiment for mitochondrial morphology, as it will ensure that all the appropriate imaging and analysis setups are in place. 1.1 To increase cellular attachment and facilitate cellular adhesion on flat optical bottom 96 well plates, add 25 µl/well of coating matrix 1:1000 in DMEM F12 by pipetting. Incubate the plates during 1 h at 37 ºC 5 % CO2. 1.2 Count SH-SY5Y using a Neubauer chamber and, after removing the coating matrix by pipetting, seed 10.000 cells/well on the coated 96 well-plate in 50 µl/well of DMEM F-12 supplemented with 10 % FBS, 2 mM Glutamine and penicillin/streptomycin. Incubate at 37 °C 5 % CO2. 1.3 Prepare for each well a mixture of 250 ng of pcDNA3.1 carrying human wild-type alphasynuclein, 0.250 µl of transfection reagent, 0.250 µl of transfection adjuvant and transfection medium up to 50 µl. Prepare a master solution with multiples of the indicated volumes according to the number of wells to transfect. 1.4 24 h after seeding the cells, remove the culture medium by manual pipetting and pour 50 µl/well of the solution prepared in step 1.3 by pipetting. Incubate at 37 °C 5 % CO2. 1.5 48 h after transfection, remove the transfection medium by pipetting and fix the cells by pipetting 25 µl/well of 4 % Paraformaldehyde (PFA) in PBS (CAUTION: paraformaldehyde is a toxic fixative, use appropriate PPE). Incubate during 5 min at R.T. 1.6 Remove the fixing solution by pipetting and wash once by pipetting 50 µl/well of PBS. 1.7 After removing the PBS by pipetting, pipet 25 µl/well of TBS with 0.05 % Tween (TBS-T) and 10 % Normal Donkey Serum (NDS). Incubate during 1 h at R.T. to block unspecific signal. 1.8 Prepare a solution of Rabbit-anti-α-synuclein antibody MJFR1 1:1000 and Mouse-antiTOMM20 antibody 1:100 in TBS-T according to the number of wells to analyse. 1.9 Remove the blocking solution by pipetting and pipet 25 µl/well of primary antibody solution prepared in step 1.8. Incubate overnight at 4 °C. 1.10 Remove the primary antibody solution and wash three times by adding and removing 50 µl/well of TBS-T by pipetting. 1.11 Prepare a solution of green secondary antibody anti-Mouse 1:1000 and red secondary antibody key anti-Rabbit 1:1000 in TBS-T according to the number of wells to analyse. 1.12 After taking out the PBS of the last wash described in step 1.10 by pipetting, pipet 25 µl/well of the secondary antibody mixture and incubate the plate during 1 h at R.T. 1.13 Remove the secondary antibody solution by pipetting and add 25 µl/well of DAPI 2 µg/ml in TBS-T by pipetting. Incubate the plate during 5 min at R.T. 1.14 Remove the DAPI solution by using a pipette and wash three times by adding and removing 50 µl/well of TBS-T with a pipette. 1.15 Pipette 80 µl/well of PBS with 0.02 % sodium azide (CAUTION: sodium azide is toxic, use appropriate PPE) and store the plate at 4 °C. 1.16 Take pictures by high-content automated fluorescence microscope or equivalent confocal imaging system equipped with a 60x objective. 1.17 Perform analysis of the TOMM20 signal of single cells (avoid any cells displaying nuclei undergoing apoptosis, necrosis or mitosis) by using Fiji: first, select and isolate the Region of Interest (ROI) by drawing around the positive or negative cell (as appropriate) and using the “Crop” function. Select the ROI by using a channel with cellular autofluorescence or a marker unrelated to the analysis in order to avoid bias in the selection of the ROI. OPTIONAL: if the image is excessively pixelated use the “Smooth” function to obtain higher edge definition. Should the smooth function be applied to an image, please apply to all subsequent images. 1.18 Then, use the “Convolve” function (Kernel mode, in Process > Filters) to reduce the background. NOTE: this is optional and usually not necessary in 5 µm tissue sections due to the thinner nature of the sections. 1.19 Activate the “Shape description” and “Limit to threshold” options on the “Set measurement” function (make sure these are activated through the analysis). 1.20 In the “Adjust” menu, select the “Threshold” function, and adjust the threshold level. The threshold settings must be maintained for all the cells of the same sample. Ensure you adequately visualize the mitochondrial network as shown in the representative images of this article, while discarding background pixels. 1.21 Use the “Analyse particles” tool on the Analyse tab. Set an appropriate size to capture the mitochondria, in this example we used size “3-Infinity”, activate “Pixel units” and select “Show: Masks” in order to visualize the result. Fiji will calculate the counts and other shape parameters. 1.22 Perform statistical analysis of the data as appropriate. In our case we used T-Test to analyse the Aspect ratio (AR) and Counts after normality testing with the D’Agostino and Pearson normality tests. Method 2: Generation of PFFs and PFF intra-cranial injections in mice Here we briefly describe the generation of the studied in situ material for the study and the intracranial injection process. This protocol is adapted from Luk et al. (25). 2.1 In order to obtain PFFs, α-Syn (5 mg/ml; Peptide) was shaken at 37 °C and 250 r.p.m. for 7 days to induce aggregation of α-synuclein. 2.2 Fibrils were then sonicated at 20 % amplitude and 0.25 cycle duty until optimal fragmentation was achieved and observed by negative stain of the samples and transmission electron microscopy. 2.3 To prepare male and female wild-type C57Bl/6 mice (3 months old) for striatal PFF injections, administer Meloxicam/Metacam (5 mg/kg) in saline solution via intraperitoneal injection and 1 ml of sterile saline solution via two 0.5 ml intradermal injection. 2.4 Induce aesthesia with 4 % Isoflurane and 0.7 % O2 in induction chamber. Shave the upper part of mouse head and gently insert the animal into the frame of the stereotactic apparatus on a heat block. 2.5 Maintain the anaesthetic plan by giving the animal inhalation anaesthesia (1-2 % Isoflurane in 0.7 % O2) through a nose mask during the whole injection process. 2.6 After cutting the skin to expose the skull and appropriate disinfection of the wound, identify the following coordinates from Bregma: - 0.5 mm anteroposterior, +/− 2.5 mm mediolateral and drill there a 0.5 mm diameter hole into the skull to expose the brain surface. 2.7 Inject 1.5 µl of PFFs by stereotactic delivery to the coordinates indicated in step 2.6 from Bregma and - 2.7 mm dorsoventral (from top brain) at a flow rate of 100 nl/min with a 32gauge Hamilton syringe. Withdrawn the syringe 5 min post injection. 2.8 Suture the wound (single surgeon stitches with 2 double knots and then a single knot), stop anaesthetic inhalation, remove the mouse from frame and let it recover in an appropriate recovery cage before returning it to the home cage. 2.9 Three months later, give the mouse 300 µl of 200 mg/ml Sodium Pentobarbital in saline solution via intraperitoneal injection. Once pain reflex is lost, make an incision on the chest, lift the ribs to expose the heart and transcardially perfuse with a 10 ml of PBS and 35 ml of 4 % Paraformaldehyde in PBS by using a 50 ml syringe connected to a 23 G butterfly needle. 2.10 Remove the brain and post-fix in 4 % PFA for 24 h at 4 °C, then remove the PFA solution and store in 70 % ethanol at 4 °C. 2.11 Put the brain into appropriate plastic embedding boxes and incubate in 95 % ethanol during one hour at R.T. Replace the embedding reagent and incubate again for 1 h. 2.12 Put the brain into appropriate plastic embedding boxes and incubate in 100 % ethanol during 1 h at R.T. Replace the embedding reagent and incubate again for 1 h. 2.13 Put the brain into appropriate plastic embedding boxes and incubate in Xylene or Xylenesubstitute during 1 h at R.T. Replace the embedding reagent and incubate again for 1 h. 2.14 Remove the Xylene or Xylene-substitute and incubate the sample in warm paraffin for 1 h. Replace the paraffin and incubate for one additional hour. 2.15 Mount the brain on the embedding boxes with warm paraffin and let it dry overnight. Cut 5 µm sections by microtome and mount them on glass slides. Method 3: Mitochondrial morphology analysis by immunohistochemistry on paraffin-embedded brain slices from PFF-injected mice 3.1 Dewax: this step is necessary to remove the paraffin and enable the rehydration of the samples. Dip the slides ten times in Xylene substitute and incubate the slides during 2 min in Xylene substitute. Dip again ten times in Xylene substitute before taking out the samples from Xylene substitute. 3.2 Rehydration: repeat the procedure described in step 3.1 with the following solutions: 100 % EtOH, 95 % EtOH, 70 % EtOH, and twice with ddH2O. 3.3 Antigen retrieval and immunostaining: start by transferring the samples to a microwavable container with ddH2O. 3.4 Warm up 100 x citrate buffer pH 6 stored at 4°C (detergents may have precipitated) and prepare 350 ml of fresh 1 x citrate buffer. Pour citrate buffer into a convenient plastic container with lid. 3.5 Put the slides into the citrate buffer container (CRITICAL: check they always stay beneath the level of the buffer) and put the lid on (CAUTION: ensure the lid is NOT completely closed otherwise the container may burst in the microwave). 3.6 Microwave at 700 W: 4 min + 5 min rest, 1.5 min + 5 min rest. Top-up citrate buffer and microwave again 1.5 min + 5 min rest, 1.5 min + 5 min rest, 1.5 min + 5min rest. 3.7 Cool down the samples in citrate buffer on ice during 20 min. Wash the sample with ddH2O. NOTE: antigen retrieval may vary according to specific antibody requirements. 3.8 Dry the samples slides by using a paper towel, without touching the tissue. Draw rectangles with pap-pen around the pieces of tissue. 3.9 Transfer all the slides to a slide immune-staining box and wash gently a couple of times with TBS + 0.05 % TWEEN (TBS-T). Check TBS-T drops stays inside the pap-pen rectangles. 3.10 Remove TBS-T from the samples by tapping on a paper towel. Gently pour 50 μl of blocking solution (10 % NDS in TBS-T) into each rectangle (without touching the tissue) and incubate for 1h at RT. NOTE: volumes may vary according to the size of the tissue, try to ensure that: i) the pap-penned area is similar across samples, and that ii) the area is fully covered by the chosen buffer volume. 3.11 Remove the blocking solution from the samples by tapping on a paper towel. Gently pour by pipetting 50 μl/rectangle of the following primary antibody mixture in TBS-T: anti-Tyrosine Hydroxylase 1:250, anti-VDAC1 1:100 and anti-pSer129 α-synuclein EP1536Y 1:2000. 3.12 Incubate overnight at 4 °C. 3.13 Wash by pipetting TBS-T on the slides and remove it by tapping on paper towel. Repeat three times. 3.14 Add by pipetting 50 μl/rectangle of secondary antibody mixture: green secondary antiChicken, red secondary anti-Mouse and far red secondary anti-Rabbit 1:1000 in TBS-T. Incubate at 37 °C during 1 h in the dark. 3.15 Wash three times with TBS-T as described in step 3.13. 3.16 Incubate with DAPI 2 µg/ml in TBS-T during 1 min. Remove DAPI solution by tapping on paper towel. Wash three times with TBS-T as described at step 3.13. 3.17 Mount a glass cover-slide on the samples by using drops/slide of mounting reagent. Gently press to eliminate bubbles and let the samples dry during 1 h at R.T. Afterwards, store at 4 °C in the dark. 3.18 Image at least 50 cells in different fields by using a structured illumination fluorescence imaging system equipped with 60x oil objective or confocal technology. 3.19 Perform analysis of the VDAC1 signal of single cells by using Fiji as indicated above. For this specific set of images, we applied a smooth filter and set the particle size as “25-infinity” on the “Analyse Particles” function from the “Analyse” menu. 3.20 Perform appropriate statistical test for your resulting analysis. REPRESENTATIVE RESULTS: In order to ensure that the appropriate imaging and analysis conditions are in place for the in situ evaluation of mitochondrial morphology in tissue, an in vitro exploration of mitochondrial morphology in response to a known modulator of mitochondrial morphology is recommended (Protocol 1). As an example, we genetically overexpressed SNCA in SH-SY5Y cells in order to induce changes in mitochondrial morphology as previously described (26). Other examples that could be used as a control to worsen mitochondria morphology would be starvation, or the use of mitochondrial activity inhibitors such as MPP+. Cells were transfected and stained for alphasynuclein (AS) so as to separate SNCA+ (AS+) and SNCA- (AS-) cells. They were also stained for TOMM20 (27) to visualize the mitochondrial network of the cells. In order to make this analysis as similar as possible to that of a 5 µm tissue section, one confocal plane was analyzed as opposed to a maximum projection of multiple planes. Morphological analysis of one confocal plane of TOMM20 revealed that both the total number of mitochondria and their aspect ratio or AR (which correlates with the elongation of the organelle) were reduced in response to SNCA overexpression (Fig. 1). We performed immunostaining for the mitochondrial protein VDAC1 in 5 µm paraffin-embedded mouse brain sections from animals injected with PFFs as described in the protocol section above. Substantia nigra pars compacta (SNc) dopaminergic neurons, which undergo degeneration in PD, were revealed through co-immunostaining with anti-Tyrosine Hydroxylase (TH) and were regionally separated from the ventral tegmental area and the substantia nigra pars lateralis. On the other hand, anti-phosphoSer129-α-synuclein (pS129) staining allowed us to discriminate cells that harbored pS129 lesions from healthy cells (pS129+ versus pS129-). SNc images of three different animals were taken and subsequent image analysis of VDAC1 staining of TH-positive neurons revealed a reduction of both mitochondrial number counts and aspect ratio between neurons bearing pS129 lesions and neurons lacking these (Fig. 2). These results indicate that the mitochondrial morphology of neurons harboring pS129 lesions is impaired in comparison to that of cells lacking pS129 lesions. While this particular experiment shows a reduction in the AR thereby highlighting a reduction in the elongation of mitochondrial together with a reduction in global counts, indicating a worsening of the mitochondrial morphology, it is important to note that the interpretation of the data should be experiment dependent. For example, a reduction in AR and counts can point to a global reduction in mitochondrial content as well as fragmentation, while a reduction in AR but an increase in global counts would point to a mitochondrial fragmentation phenotype. Therefore, it is important to interpret the data in the context of both measures. FIGURE AND TABLE LEGENDS: Figure 1: Mitochondrial morphology in a SNCA-overexpressing in vitro model. Co-immunostaining for TOMM20 (green), α-synuclein (AS, red) and DAPI (blue) on SNCA-overexpressing and not overexpressing (AS+ and AS-, respectively) cells (A). Detail of a AScell (B), and of a AS+ cell (C). Black and white images in panels B and C represent the masks of the TOMM20 signal after applying the Fiji function described in the Protocol section. This mask enables the quantification of the shape of the resulting structures. Mitochondrial counts and Aspect Ratio (AR) values of ASand AS+ cells (N=25 cells per condition) were quantified and represented as individual values as Table 1 Name of Material/ Equipment Company Catalog Number Comments/Description Alexa fluor 488/594-Donkey anti-Mouse Invitrogen A21202; A21203 green/red dye-Donkey antiMouse Alexa fluor 594/647-Donkey anti-Rabbit Invitrogen A21207 A31573 red/far red dye-Donkey antiRabbit AlexaFluor 488-Donkey anti-Chicken Jackson ImmunoResearch 703-545-155 green dye-Donkey anti-Chicken Anti-α-synuclein antibody MJFR1 (Rabbit) Abcam ab138501 Anti-PSer129 α-synuclein EP1536Y (Rabbit) antibody Abcam ab51253 Anti-Tyrosine Hydroxylase (Chicken) antibody Abcam ab76442 Anti-TOM 20 (Mouse) antibody Santa Cruz sc-17764 Anti-VDAC1 (Mouse) antibody Santa Cruz sc-390996 Citrate buffer 100X stock: 120mM citrate buffer, 5% Tween in water (pH 6) Home-made EVOS M7000 Imaging System ThermoFisher Scientific High-content automated fluorescence microscope 4',6-diamidino-2-fenilindol, dihidrocloruro (DAPI) Invitrogen D1306 D-MEM F12 Gibco A321331020 Fetal Bovine Serum Gibco 10270106 Flat optical bottom 96 well plates Greiner 675090 FluorSave Reagent Millipore 345789-20ML Mounting reagent Glutamine 200mM Gibco 25030-024 32-gauge Hamilton syringe Hamilton 7632-01 Lipofectamine and Plus Reagent Invitrogen 11668-019; 11514-015 Transfection reagent and transfection adjuvant Matrigel Corning 354230 Coating matrix Microtome ThermoFisher Scientific Normal Donkey Serum Gibco PCN5000 ImmEdge Hydrophobic Barrier Pen Vector Laboratories H-4000 PAP-pen Penicillin/Streptomycin solution Gibco 15140-122 Opti-MEM Gibco 31985070 Transfection medium PCDNA4 plasmid (backbone) Addgene 41036 SH-SY5Y cells/well ATCC HTB-11 Xylene substitute Labbox 22L36504 Zeiss Axio Imager Apotome 2 Carl Zeiss Structured illumination fluorescence imaging system