Hello,
In a co-culture of astrocytes and neurons, I would like to only inhibit the lysosomal function of the neurons and/or the astrocytes independently of each other (i.e., to not inhibit lysosomal activity of both cell types if I add a compound into my coculture). .My questions thus are:
1) Are there any specific inhibitors for one cell or the other that I could use? If possible I would like to timely inhibit lysosomal activity on one of the cell types while in co-culture, but I am not familiar enough to know if this is possible or not. If the answer is no:
2) Could I inhibit the lysosomal function of one or the other prior to co-culturing them or would this affect the normal properties of the coculture? This is my less than ideal alternative, but I was wondering if it still could be done without serious damage to the cell.
Thanks in advance!
I have been pondering this question for a while now, but I believe there is no simple solution, unfortunately. There are, to my knowledge, no specific inhibitors. The astrocytes and the neurons are (I assume) from the same species and the basic protein machinery will be identical. Even if you would be able to co-culture, say, rat neurons on mice astrocytes, there are still no species-selective inhibitors, at least to my knowledge; the key lysosomal proteins are just too similar.
If you go for inhibition of one cell type before bringing them together in co-culture, you might run into problems pretty fast. Inhibiting the neurons before co-culture is most likely impossible, as it will mess up their development. For astrocytes, it is more feasible to inhibit them right before co-culture, but you have to assume that they will either (a) die before the neurons mature, (b) come back to baseline from the inhibition before the neurons mature, as you will not be able to continue the drug treatment after you introduce the neurons, or (c) the drug will affect the neurons, too, since most inhibitors can also detach again from their target and find a new one (potentially in the neurons). In conclusion, I'm not optimistic about a culture like that developing reliably.
So the only feasible approach I see would be to genetically introduce a system you can switch on/off at will. There are a few ways one could go about it:
1) Target the neurons (or astrocytes) with specific transfection agents. Semliki Forest Virus, for example, is pretty specific in targeting neurons. You could use it to knock down a key gene of lysosomes through RNAi. Along the same lines: maybe you can source a rat or mouse line with a conditional knock-out system that you can induce in culture?
2) Transfect the astrocytes (before you introduce the neurons) with an RNAi construct to knock down a key gene of lysosomes, but make the system inducible. You could then give the stimulus for expression after you introduce the neurons.
These approaches are rather cumbersome, and might require extensive optimisation, however. But it is the only thing I can come up with right now. It is a very interesting problem/question, and I hope you find a solution that works!
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