Investigations into the effects of serine proteases in modulating long lasting depression of synaptic transmission

Long-term depression is a long-lasting decrease in signalling between neurons in the central nervous system. It can be elicited via a number of mechanisms, including NMDA receptors and mGLuR receptors. NMDA-dependent LTD is caused by the endocytosis of AMPA receptors in response to stimuli, whereas mGLuR-dependent LTD acts via the action of kinases. Recent studies have demonstrated a novel form of LTD, which is dependent on serine protease action. Molecular changes in protein expression also accompany the generation of this new type of LTD, although the exact mechanism has not been determined. The aims of this current study are to determine the mechanism by which subtilisin could modulate LTD, and whether this effect was solely dependent on proteolytic action, or other factors. Subtilisin-mediated LTD was elicited in hippocampal slices of mice as an experimental model, and each slice was retained at the end of every experiment to enable protein levels to be analysed via immunoblotting. Initial experiments were carried out to evaluate the relationship between subtilisin activity and metalloprotease action, based on previous studies that demonstrated subtilisin proteolysis of the synaptic protein VAMP-1, a known target of the metalloprotease tetanus toxin. The putative link between these two proteases was tested by addition of zinc to the perfusing solution, which promotes the activity of zinc-based metalloproteases. Subtilisin however was completely unaffected by this change to extracellular levels of zinc. Experiments were also conducted in the presence of the metal ion chelators EDTA and captopril to determine the contribution of metal ions to subtilisin action. Addition of the general metal chelator EDTA failed to decrease subtilisin action, and the zinc-specific chelator captopril likewise did not display inhibitory action against subtilisin. Attempts were also made to produce a direct comparison between the action of subtilisin and tetanus toxin, however, preliminary experiments were unsuccessful as the tetanus toxin used in the experiment was unable to cause VAMP-1 proteolysis. The possibility that subtilisin action was solely dependent on their inherent serine protease function was also explored using the general serine protease inhibitor phenylmethanesulfonylfluoride (PMSF). The use of PMSF could provide a better indication of the link between LTD and subtilisin proteolysis, and also shed light on the order of protein degradation. Perfusion of PMSF-inactivated subtilisin prevented the proteolysis of proteins, and also prevented the generation of LTD and abolished the proteolytic activity of subtilisin. These results indicate a close link between the LTD- 2 inducing and proteolytic effects of subtilisin, and in conjunction with the metalloprotease experiments, suggest a serine protease basis for subtilisin-mediated LTD. These results suggested that subtilisin action was not based on metalloprotease degradation of VAMP-1 alone, but on a more general proteolytic effect involving other protein targets such as the netrin receptor Unc5H3 and the cytoskeletal protein actin. The specific role of the Unc5H3 and VAMP-1 proteins in the subtilisin process was studied using protein- specific antibodies to prevent proteolysis. Preincubation of hippocampal slices with VAMP-1 proteins did not prevent the proteolytic effects of subtilisin, nor could it prevent the generation of LTD. Preincubation of hippocampal slices with VAMP-1 antibody in the presence of either the cell permeabiliser triton, or the pore-forming chemical streptolysin also had no inhibitory effect on subtilisin action. Hippocampal slices were also preincubated with Unc5H3 antibodies to determine the importance of this protein to the activity of subtilisin; however, this did not change the proteolytic or electrophysiological effects of subtilisin. Addition of either triton or streptolysin to the preincubation solution containing Unc5H3 antibodies failed to prevent the effects of subtilisin on the hippocampal slice, a result identical to those obtained with VAMP-1 antibody preincubations. The importance of actin proteolysis in mediating subtilisin action was also assessed by addition of the actin stabiliser jasplakinolide to the aCSF used in perfusions to prevent actin degradation. Jasplakinolide was unable to prevent the onset of subtilisin-mediated LTD or its associated degradation of actin. In order to verify whether the effects of subtilisin represented a novel form of LTD, the LTD elicited by subtilisin was compared to that of more established methods of eliciting LTD, such as the mGluR agonist DHPG. Slices exposed to DHPG failed to elicit LTD, however, the GABA mimetic ethylenediamine (EDA) appeared to be a potent inducer of LTD. EDA produced a significantly smaller LTD effect in comparison to subtilisin, which was not accompanied by degradation of any proteins associated with subtilisin perfusion. It would be of interest to know whether other serine proteases could cause this LTD effect, and based on previous studies, it is known that members of the S8A subfamily and the S1A subfamily of serine proteases are capable of causing the same effects as subtilisin. To date much work has been performed on subtilisin, and other groups S8A serine proteases such as Proteinase-K. In comparison, very little is known about of the mechanism utilised by group S1A serine proteases in causing LTD. In order to gain a greater understanding of The possibility that other serine proteases could generate LTD was also investigated as part of this study, with particular emphasis on members of the S8A (to which subtilisin belongs) and S1A subfamily of serine proteases, which are biochemically similar to 3 members of the S8A subfamily. Previous studies have shown that proteinase-K and cadeprin, both members of the S8A, as well as α-chymotrypsin, a member of the S1A subfamily, can replicate this LTD effect. By studying the impact of α-chymotrypsin on fEPSP and protein degradation, it was hoped that this would provide a much more balanced view on the mechanisms behind the LTD-inducing effects of serine proteases. Previous experiments with subtilisin had ruled out the action of mGluRs or the role of electrically-stimulated LTD as the basis for subtilisin-mediated LTD. For this study these two factors were investigated using α-chymotrypsin in order to discover the different mechanisms utilised by these two serine proteases. Electrically-elicited LTD is known to be sensitive to the action of protein phosphatases, and in the light of this knowledge, experiments were conducted using the phosphatase inhibitors phenylarsine oxide (PAO) and sodium orthovanadate. Sodium orthovanadate did not affect the progression of α- chymotrypsin LTD; however, PAO did significantly decrease the magnitude of this type of LTD. This suggests a possible role for tyrosine kinase in this process, although more experiments would be necessary to provide a definite link as sodium orthovanadate had no effect on the action of chymotrypsin. The involvement of mGLuR in α-chymotrypsin LTD was tested using the p38 MAPK inhibitor SB203580. Perfusion of this chemical did not result in a noticeable decrease in α-chymotrypsin activities, and taken together with previous experiments in subtilisin, suggests that serine protease-mediated LTD is not solely dependent on these two pathways for their neuronal depressive actions. The results presented here strongly suggest that LTD caused by serine proteases such as subtilisin and α-chymotrypsin do not involve metalloprotease functions. Instead, LTD is closely linked with the proteolysis of a select number of proteins that are cleaved by the endogenous proteolytic functions of these serine proteases. Furthermore, serine protease- mediated LTD is not simply restricted to subtilisin alone, but can involve other members of the S8A subfamily of serine proteases such as α-chymotrypsin. Information gathered over these investigations also suggests the involvement of mechanisms utilised by other forms of LTD, but their overall action depends on categorically different mechanisms.