Mitochondria play a crucial role in Parkinson’s disease (PD) pathogenesis but also other neurological conditions, as they are central to cellular energy production, calcium regulation, and apoptosis. Dysfunction of these organelles has been shown to be involved in the complex process leading to the death of dopaminergic neurons that are characteristic for PD. Several neurotoxins, including 6-OHDA, rotenone, antimycin, and MPP+ are commonly used to induce mitochondrial impairment.
At Scantox, several assays combining different lesions are set up. Cell models as well as mitochondrial marker are available:
- 6-OHDA, a neurotoxin that selectively targets dopaminergic neurons, disrupts mitochondrial function by inducing oxidative stress and impairing complex I activity. This leads to ATP depletion and the generation of reactive oxygen species (ROS), ultimately contributing to neuronal death.
- Rotenone, a potent inhibitor of mitochondrial complex I, interferes with electron transport, resulting in a decreased ATP production and increased ROS generation. This disruption of mitochondrial function mirrors the mitochondrial dysfunction observed in PD patients and contributes to dopaminergic neuron degeneration.
- Antimycin, an inhibitor of mitochondrial complex III, disrupts electron flow within the respiratory chain, leading to mitochondrial dysfunction and ROS production. This impairment of mitochondrial activity contributes to neuronal damage and has been implicated in PD pathogenesis.
- MPP+, a metabolite of MPTP, selectively targets dopaminergic neurons by inhibiting mitochondrial complex I, disrupting ATP production, and inducing oxidative stress. This leads to mitochondrial dysfunction and neuronal death, mimicking key aspects of PD pathology.
Mitochondrial activity can be measured using various techniques, including fluorescent probes such as:
- TMRM (tetramethylrhodamine methyl ester) and Mitotracker selectively accumulate in active mitochondria, allowing to assess mitochondrial membrane potential and function in live cells even over time by utilizing the IncuCyte® live-cell imaging system.
- DCFDA and MitoSOX are utilized to assess oxidative stress levels and mitochondria-specific ROS production, respectively. DCFDA measures overall cellular ROS levels, while MitoSOX specifically targets mitochondria, providing insights into mitochondrial oxidative stress.
- JC-1 is commonly used to evaluate mitochondrial membrane potential changes associated with PD-specific neurotoxins such as MPP+. JC-1 allows for the quantification of mitochondrial membrane potential alterations, contributing to the understanding of mitochondrial dysfunction in PD pathogenesis.
All assays can be performed on a variety of cell types:
- SH-SY5Y cells, a human neuroblastoma cell line often used as model for dopaminergic neurons.
- Primary rodent cortical or hippocampal neurons, offering insights into general neuronal responses.
- Primary rodent neurons from the ventral mesencephalon, focusing on dopaminergic neurons directly affected by PD
- iPSC-derived neurons, induced pluripotent stem cell-derived neurons providing a human-specific context and essential for translating findings into clinical applications.
At Scantox, these assays are frequently performed, and we are happy to adapt the workflow based on the specific needs of your next research project.
Figure 1: Mitochondrial activity of primary cortical mouse neurons assessed using TMRM (tetramethylrhodamine methyl ester). A: Total integrated intensity of TMRM over time. Data are presented as data points + standard error of the mean (SEM; n=8 per group). B: Area under the curve (AUC) of total integrated intensity of TMRM for 24 h. Data are presented as bar graphs with standard error of the mean (SEM; n=6 per group). MK-801 served as a reference item. For statistical analysis One-way ANOVA followed by Dunnett’s post hoc test versus MPP+ lesion was used. ***p<0.001.
Figure 2: Representative videos of TMRM fluorescence for vehicle (left) and MPP+ (right)-lesioned primary cortical mouse neurons.
We are happy to evaluate the efficacy of your compound in these in vitro PD models!