Abstract
The current study investigates the neurotrophic effects of Clostridium botulinum C3 transferase (C3bot) on highly purified, glia-free, GABAergic, and glutamatergic neurons. Incubation with nanomolar concentrations of C3bot promotes dendrite formation as well as dendritic and axonal outgrowth in rat GABAergic neurons. A comparison of C3bot effects on sorted mouse GABAergic and glutamatergic neurons obtained from newly established NexCre;Ai9xVGAT Venus mice revealed a higher sensitivity of GABAergic cells to axonotrophic and dendritic effects of C3bot in terms of process length and branch formation. Protein biochemical analysis of known C3bot binding partners revealed comparable amounts of β1 integrin in both cell types but a higher expression of vimentin in GABAergic neurons. Accordingly, binding of C3bot to GABAergic neurons was stronger than binding to glutamatergic neurons. A combinatory treatment of glutamatergic neurons with C3bot and vimentin raised the amount of bound C3bot to levels comparable to the ones in GABAergic neurons, thereby confirming the specificity of effects. Overall, different surface vimentin levels between GABAergic and glutamatergic neurons exist that mediate neurotrophic C3bot effects.
Highlights
Monomeric GTPases of the Rho family are important cellular switches that regulate neuronal morphology
We looked at the morphological effects elicited by medium application of Clostridium botulinum C3 transferase (C3bot) on neocortical/hippocampal neurons prepared from early postnatal brains
To see whether the effect of an increased process formation resulted from enhanced dendrite formation in C3bot-treated neurons we stained the neurons for the dendritic marker microtubuleassociated protein 2 (Map2) and counted the number of dendritic processes
Summary
Monomeric GTPases of the Rho family are important cellular switches that regulate neuronal morphology. RhoA usually functions as a negative regulator of axon outgrowth and together with RhoB and C can be inhibited by Clostridium botulinum C3 exoenzyme (C3bot) that represents the prototype of bacterial ADP-ribosyltransferases (Aktories and Frevert, 1987) For this reason, C3bot and derivatives have been extensively used as tools to investigate neurite outgrowth and to foster neuronal plasticity for the potential treatment of CNS traumas like spinal cord injuries (SCI). Astroglial upregulation and subsequent vimentin release in vivo has been demonstrated by other groups in the intact and injured CNS (Teshigawara et al, 2013; Shigyo and Tohda, 2016) In this context, we were able to show that exosomes obtained from cultured scratch-injured wild type but not vim−/− astrocytes enhanced binding of C3bot to synaptosomes from the brain and spinal cord (Adolf et al, 2019). The regeneration-promoting CNS effects of C3bot or C3bot-derived peptides on axon regeneration in vivo in defined neuron types such as the primarily glutamatergic retinal ganglion cell axons in the optic nerve (Bertrand et al, 2007; Hu et al, 2007) as well as glutamatergic corticospinal or serotonergic descending brainstem fibers (Boato et al, 2010) display the effects in a glial environment
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