measurement-mitochondrial-membrane-potential-vivo-using-genetically

Measurement of Mitochondrial Membrane Potential <em>In Vivo</em> using a Genetically Encoded Voltage Indicator

Mitochondrial membrane potential (MMP, &#916;&#936;m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen ...

the First Affiliated Hospital of Zhengzhou University

Zhengzhou University

The temperature of a living cell is a crucial parameter for cellular events, such as cell division, gene expressions, enzyme activities and metabolism. We previously developed a quantifiable mitochondrial thermometry 1.0 based on rhodamine B methyl ester (RhB-ME) and rhodamine 800 (Rh800), and the theory for mitochondrial thermogenesis. Given that the synthesized RhB-ME is not readily available, thus, a convenient mitochondrial thermometry 2.0 based on tetra-methyl rhodamine methyl ester (TMRM) and Rh800 for the thermogenic study of brown adipocyte was further evolved. The fluorescence of TMRM is more sensitive (∼1.4 times) to temperature than that of RhB-ME, then the TMRM-based mito-thermometry 2.0 was validated and used for the qualitatively dynamic profiles for mitochondrial thermogenic responses and mitochondrial membrane potential in living cells simultaneously. Furthermore, our results demonstrated that the heterogenous thermogenesis evoked by β3 adrenoceptor agonist only used overall up to ∼46% of the thermogenic capacity evoked by CCCP stimulation. On the other hand, the results demonstrated that the maximum thermogenesis evoked by NE and oligomycin A used up to ∼79% of the thermogenic capacity, which suggested the maximum thermogenic capacity under physiological conditions by inhibiting the proton-ATPase function of the mitochondrial complex V, such as under the cold activation of sympathetic nerve and the co-release of sympathetic transmitters.

A sensitive mitochondrial thermometry 2.0 and the availability of thermogenic capacity of brown adipocyte.

Parkinson's disease (PD) is a mitochondria-related neurodegenerative disease characterized by locomotor deficits and loss of dopaminergic (DA) neurons in the  (SNc). Majority of PD research primarily focused on neuronal dysfunction, while the roles of astrocytes and their mitochondria remain largely unexplored. To bridge the gap and investigate the roles of astrocytic mitochondria in PD progression, we constructed a specialized optogenetic tool, mitochondrial-targeted anion channelrhodopsin, to manipulate mitochondrial membrane potential in astrocytes. Utilizing this tool, the depolarization of astrocytic mitochondria within the SNc in vivo led to the accumulation of γ-aminobutyric acid (GABA) and glutamate in SNc, subsequently resulting in excitatory/inhibitory imbalance and locomotor deficits. Consequently, in vivo calcium imaging and interventions of neurotransmitter antagonists demonstrated that GABA accumulation mediated movement deficits of mice. Furthermore, 1 h/day intermittent astrocytic mitochondrial depolarization for 2 weeks triggered spontaneous locomotor dysfunction, α-synuclein aggregation, and the loss of DA neurons, suggesting that astrocytic mitochondrial depolarization was sufficient to induce a PD-like phenotype. In summary, our findings suggest the maintenance of proper astrocytic mitochondrial function and the reinstatement of a balanced neurotransmitter profile may provide a new angle for mitigating neuronal dysfunction during the initial phases of PD.

Neurotransmitter accumulation and Parkinson's disease-like phenotype caused by anion channelrhodopsin opto-controlled astrocytic mitochondrial depolarization in substantia nigra pars compacta.

Mitochondrial membrane potential (MMP) plays a crucial role in the function of cells and organelles, involving various cellular physiological processes, including energy production, formation of reactive oxygen species (ROS), unfolded protein stress, and cell survival. Currently, there is a lack of genetically encoded fluorescence indicators (GEVIs) for MMP. In our screening of various GEVIs for their potential monitoring MMP, the Accelerated Sensor of Action Potentials (ASAP) demonstrated optimal performance in targeting mitochondria and sensitivity to depolarization in multiple cell types. However, mitochondrial ASAPs also displayed sensitivity to ROS in cardiomyocytes. Therefore, two ASAP mutants resistant to ROS were generated. A double mutant ASAP3-ST exhibited the highest voltage sensitivity but weaker fluorescence. Overall, four GEVIs capable of targeting mitochondria were obtained and named mitochondrial potential indicators 1-4 (MPI-1-4). In vivo, fiber photometry experiments utilizing MPI-2 revealed a mitochondrial depolarization during isoflurane-induced narcosis in the M2 cortex.

Development and Application of a Mitochondrial Genetically Encoded Voltage Indicator in Narcosis.

Pluripotent stem cells were generated through the electroporation of episomal plasmids, containing crucial reprogramming factors, into skin fibroblasts extracted from a female Alzheimer's patient harboring the PSEN1 709 T > C (p.Phe237Leu) heterozygous mutation. The pluripotent stem cells exhibit a normal karyotype and express pivotal stem cell markers including TRA-1-60, Nanog, SOX2, and OCT4. Furthermore, their capacity to differentiate into the three germ layers in in vivo teratoma experiments has been substantiated. The pluripotent stem cell line can serve as a cellular model for Alzheimer's disease, offering significant value in elucidating the pathogenesis and therapeutic strategies of the disease.

Generation of an induced pluripotent stem cell line (CSBZZUi001-A) from a female Alzheimer's patient carrying the PSEN1 709 T > C heterozygous mutation.

We recruited a healthy 44-year-old female and obtained her skin fibroblasts. Subsequently, the induced pluripotent stem cell line was successfully established using non-integrated reprogramming technology. The cell line had a normal karyotype and has been confirmed to have good pluripotency through the detection of pluripotency markers and detection of teratoma formation. This cell line can serve as an effective control for studying the cellular pathological mechanisms of other specific mutations.

Construction of induced pluripotent stem cell line (CSBZZUi002-A) from the fibroblast cells of a healthy female.

Aging is an intricate process involving interactions among multiple factors, which is one of the main risks for chronic diseases, including Alzheimer's disease (AD). As a member of cysteine protease, cathepsin S (CTSS) has been implicated in inflammation across various diseases. Here, we investigated the role of neuronal CTSS in aging and AD started by examining CTSS expression in hippocampus neurons of aging mice and identified a significant increase, which was negatively correlated with recognition abilities. Concurrently, we observed an elevation of CTSS concentration in the serum of elderly people. Transcriptome and fluorescence-activated cell sorting (FACS) results revealed that CTSS overexpression in neurons aggravated brain inflammatory milieu with microglia activation to M1 pro-inflammatory phenotype, activation of chemokine C-X3-C-motif ligand 1 (CX3CL1)-chemokine C-X3-C-motif receptor 1 (CX3CR1) axis and janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway. As CX3CL1 is secreted by neurons and acts on the CX3CR1 in microglia, our results revealed for the first time the role of neuron CTSS in neuron-microglia "crosstalk." Besides, we observed elevated CTSS expression in multiple brain regions of AD patients, including the hippocampus. Utilizing CTSS selective inhibitor, LY3000328, rescued AD-related pathological features in APP/PS1 mice. We further noticed that neuronal CTSS overexpression increased cathepsin B (CTSB) activity, but decreased cathepsin L (CTSL) activity in microglia. Overall, we provide evidence that CTSS can be used as an aging biomarker and plays regulatory roles through modulating neuroinflammation and recognition in aging and AD process.

Neuronal cathepsin S increases neuroinflammation and causes cognitive decline via CX3CL1-CX3CR1 axis and JAK2-STAT3 pathway in aging and Alzheimer's disease.

Article	Year
A sensitive mitochondrial thermometry 2.0 and the availability of thermogenic capacity of brown adipocyte. Frontiers in physiology\| PubMed ID: 36091398	2022
Neurotransmitter accumulation and Parkinson's disease-like phenotype caused by anion channelrhodopsin opto-controlled astrocytic mitochondrial depolarization in substantia nigra pars compact... MedComm\| PubMed ID: 38756440	2024
Development and Application of a Mitochondrial Genetically Encoded Voltage Indicator in Narcosis. Neuroscience bulletin\| PubMed ID: 38829505	2024
Generation of an induced pluripotent stem cell line (CSBZZUi001-A) from a female Alzheimer's patient carrying the PSEN1 709 T > C heterozygous mutation. Stem cell research\| PubMed ID: 38968760	2024
Construction of induced pluripotent stem cell line (CSBZZUi002-A) from the fibroblast cells of a healthy female. Stem cell research\| PubMed ID: 39442281	2024
Neuronal cathepsin S increases neuroinflammation and causes cognitive decline via CX3CL1-CX3CR1 axis and JAK2-STAT3 pathway in aging and Alzheimer's disease. Aging cell\| PubMed ID: 39453382	2025

Dian-Dian Wang

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