Nature Sub-Journal: New Technology!

Click on the blue letters to pay attention to our neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, nutritional lateral sclerosis and other neurodegenerative diseases that have typical neuronal dysfunction and death
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At present, there are some fluorescent dyes and indicators for selectively marking neuronal death, but the permeability of these dyes in tissues is different, so quantification is difficult
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In addition, dyes are also more toxic
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Therefore, it is challenging to accurately mark neurons that are alive, dead, or dying
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On September 6, 2021, Steven Finkbeiner's research team at the Gladstone Institute in San Francisco modified the gene-encoded calcium ion indicator to develop a fluorescent probe GEDI that marks dead cells so that it can be located in the cytosol.
It will not respond to neuron activation, but will respond to changes in calcium ions caused by the time of neuron death, so as to mark dead neurons
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Calcium-measuring organelle-entrapped protein indicators (CEPIAer) are indicators that can detect changes in calcium ions in the endoplasmic reticulum or mitochondria of organelles
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The researchers modified the CEPIA mutant so that it was originally located on the organelle membrane and then only located in the cytoplasm after modification
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In vitro rat neurons were infected with GCaMP6f or CEPIA with red fluorescence (RGEDI).
Under electrical stimulation, the fluorescence intensity of GCaMP6f increased sharply, but the fluorescence of RGEDI did not change
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But in the process of neuron death caused by sodium azide, the fluorescence intensity of GCaMP6f and RGEDI both increased instantaneously
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This indicates that RGEDI can mark dead neurons
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In addition, in the process of neuron death, the RGEDI labeling occurs earlier than the dead neuron's structural changes (lysis), and also earlier than the appearance of apoptosis markers, which further indicates that GEDI can specifically label the early events of cell death
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Glutamate is a key neurotransmitter in the brain.
Excessive presence of glutamate can cause neuronal death
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The researchers' clever neuron promoter, green fluorescent protein, and RGEDI are connected together to construct the viral vector hSyn1:RGEDI-P2a-EGFP, which can simultaneously label dead and alive neurons
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The neurons in vitro are transfected with hSyn1:RGEDI-P2a-EGFP and given excessive glutamate, and the whole process of neuronal death and lysis can be observed dynamically and in real time
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Does RGEDI have the potential to mark neuronal death events in neurodegenerative diseases? The isolated neurons were transfected with α-synuclein and TDP43 protein to simulate the pathological state of Parkinson's and lateral sclerosis in vitro.
GEDI can efficiently label the dead neurons in the above model, further confirming its practicability
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However, behind the perfection of GEDI probes, there are also huge flaws-the inability to achieve in vivo labeling
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Therefore, the researchers expanded the members of the GEDI probe family and developed the fluorescent probe GC150 that can label dead neurons in vivo, which has been well verified on the zebrafish model
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They also developed a type of GEDI that is localized to the nucleus
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GCaMP6-150 is a type of indicator that senses changes in endoplasmic reticulum calcium ions.
After removing the endoplasmic reticulum signal peptide, the researchers added P2a peptide and mApple fluorescent protein to obtain GC150, which greatly improved the ability to sense cell death
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In general, this article modified the gene-encoded calcium ion indicator so that it can be located in the cytosol.
This will not respond to neuron activation, but will change the calcium ion caused by the time of neuron death.
In response, the dead neurons are marked
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[References] 1.
https://doi.
org/10.
1038/s41467-021-25549-9 The pictures in the article are from the references