Name: Video: Mitochondrial Morphology Analysis by Immunohistochemistry on Paraffin-Embedded Brain Slices from α-Synuclein Pre-Formed Fibrils (PFF)-Injected Mice
Uploaded: 2023-06-23T14:50:00
Duration: 4 min 21 s
Description: 237 Views. Mitochondrial Morphology Analysis by Immunohistochemistry on Paraffin-Embedded Brain Slices from α-Synuclein Pre-Formed Fibrils (PFF)-Injected Mice

mitochondrial morphology analysis by immunohistochemistry on paraffin-embedded brain slices from &#945;-synuclein pre-formed fibrils (pff)-injected mice

An In Situ Immunostaining Protocol for Parkinson's Disease Study

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 the ATP used by the brain is produced by the mitochondria1. Therefore, it is not surprising that mitochondrial dysfunction is particularly harmful to neurons. In fact, mitochondrial function impairments play an important role in several neurological diseases, including neurodegenerative conditions, such as Parkinson&#39;s Disease (PD) and Alzheimer&#39;s Disease (AD)2,3.
Multiple genes are unequivocally linked to PD-encoding proteins that are relevant for mitochondrial function and homeostasis, such as Parkin4,5,6, PTEN-induced kinase 1 (PINK1)7,8 and DJ-19. Further evidence for a role for mitochondrial dysfunction in PD is that treatments with inhibitors of Complex I of the mitochondrial electron transport chain (such as Rotenone and MPTP) recapitulate several aspects of PD in vitro and in vivo10. However, it is important to state that many pathological processes may drive 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 among the most studied (reviewed in11,12,13 and).
Mitochondria are heterogeneous in 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 function14; in fact, mitochondrial networks are extremely dynamic, undergoing frequent processes of fission, fusion, and mitophagy in order to meet the needs of the cells and to respond to environmental cues15,16. 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 hyperfused mitochondria17. On the other hand, a variety of human diseases present aberrant mitochondrial morphology, including mitochondrial fragmentation or excessive mitochondrial fusion, which have harmful effects on mitochondrial function (reviewed in18). In the context of PD, we and others have previously shown that abnormal mitochondrial shape correlates with dysfunction in response to &#945;-synuclein aggregates19. While mitochondrial morphology has been extensively studied in vitro both in the context of PD and other diseases20,21,22, protocols for the evaluation of mitochondrial morphology from in vivo sections are lacking. This makes the in vivo study of mitochondria in the context of diseases such as PD highly dependent on transgenic animals23 or the evaluation of midbrain extracts that cannot provide cellular resolution.
Here, a protocol is presented to study the mitochondrial morphology in situ as an indicator of their functional status and health, based on immunostaining of the mitochondrial protein VDAC124 followed by image analysis in paraffin-embedded tissue sections. We also show the results of this protocol in in vitro and in vivo PD models: neuroblastoma cells overexpressing SNCA (Synuclein Alpha) and brain tissue from mice subjected to intracranial injection of &#945;-synuclein Pre-Formed Fibrils (PFFs). Co-immunostaining with an antibody against &#945;-synuclein (in cells) or phosphoSer129- &#945;-synuclein pS129 (in mouse brains) allowed us to identify cells with aggregate protein pathology (overexpressed &#945;-synuclein and &#945;-synuclein fibrils, respectively) in the samples, while negative cells served as a non-pathological control within the same samples. Through this analysis and the data described here, a reduced aspect ratio was observed, indicating the fragmentation of mitochondria in cells overexpressing SNCA or presenting pS129 lesions.

Author Spotlight: Establishing a New Fluorescence-Based Protocol for In Vivo Mitochondrial Morphology Analysis in Parkinson's Disease

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

Mitochondrial Morphology Analysis by Immunohistochemistry on Paraffin-Embedded Brain Slices from &#945;-Synuclein Pre-Formed Fibrils (PFF)-Injected Mice

mitochondrial-morphology-analysis-immunohistochemistry-on-paraffin

Research

JoVE Journal

Neuroscience

237 Views. Mitochondrial Morphology Analysis by Immunohistochemistry on Paraffin-Embedded Brain Slices from α-Synuclein Pre-Formed Fibrils (PFF)-Injected Mice

Video: Mitochondrial Morphology Analysis by Immunohistochemistry on Paraffin-Embedded Brain Slices from α-Synuclein Pre-Formed Fibrils (PFF)-Injected Mice

Mitochondria play a central role in the energy metabolism of cells, and their function is especially important for neurons due to their high energy 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, and 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 because it can detect subtle differences in mitochondrial counts and shape induced by aggregates of &#945;-synuclein, an aggregation-prone protein heavily involved in the pathology of Parkinson's disease. This method allows one to report that substantia nigra pars compacta dopaminergic neurons harboring pS129 lesions show mitochondrial fragmentation (as suggested by their reduced Aspect Ratio, AR) compared to their healthy neighboring neurons in a pre-formed fibril intracranial injection Parkinson model.

This study presents a method to analyze the morphology of mitochondria based on immunostaining and image analysis in mouse brain tissue in situ. It also describes how this allows one to detect changes in mitochondrial morphology induced by protein aggregation in Parkinson's disease models.

Mitochondria play a central role in the energy metabolism of cells, and their function is especially important for neurons due to their high energy ...