Silencing synapses

Abstract

The degeneration of pre-synaptic boutons in the stratum radiatum of the dorsal hippocampus is one of earliest components of neurodegeneration in several models of murine prion disease. We recently showed that blockade of synaptic transmission by infusion of botulinum neurotoxin A (BoNT/A) into the hippocampus several weeks prior to the onset of degeneration, had no detectable impact on the extent of the synaptic degeneration.¹ We elaborate here on the rationale for these experiments and highlight why we believe that this negative result is interesting and important. We also discuss new observations that might provide insights into the molecular events that underlie synapse degeneration.

The idea that synapse loss may be the earliest neurodegenerative event in a protein misfolding disease was first suggested on the basis of studies in Alzheimer disease now some 30 y ago.² However, establishing that synapse degeneration is indeed the first neurodegenerative event and whether it is the pre-synaptic or post-synaptic element that degenerates first has only been established in a few instances: in particular in circumstances where the neuroanatomical circuitry permits a clear assay of the integrity of both the synapses and the cell soma of origin. In murine prion disease, including our own work on the ME7 model, it is clear that the degeneration of the presynaptic boutons from CA3 axons terminating on the spines of CA1 cells precedes degeneration of the cell soma.^3,4 Biochemical analysis of the changes in synapse related proteins in the hippocampus during the evolution of ME7 prion disease indicated that several synaptic vesicle proteins, such as cysteine string protein (CSP)-α, VAMP-2 and synapsin were reduced prior to the loss of post-synaptic density (PSD) related proteins.⁵ Electron microscopy studies showed that the degeneration of the presynaptic bouton was associated with the dendritic spine PSD curving around and enveloping the degenerating element in the absence of involvement of glial processes (Fig. 1). In another neurodegenerative disease, amyotrophic lateral sclerosis, the synapses at the neuromuscular junction degenerate prior to the cell body.⁶ Studying these processes of early synaptic degeneration is of importance since it has been shown that reduction of synaptic density correlates well with functional impairments in neurodegenerative conditions.^2,7 Thus, understanding and preventing synapse death is of obvious importance from a therapeutic point of view.

Figure 1. Electron micrograph to illustrate a presynaptic terminal in the stratum radiatum undergoing degeneration (indicated by an arrow) during disease progression in the ME7 model of murine prion disease. An healthy, nearby synaptic terminal is indicated by an arrowhead. Scale bar = 500 nm.

There is evidence to suggest that neuronal electrical activity may play a role in synapse degeneration in several protein misfolding diseases.^8-10 In particular, studies have suggested that synaptic activity drives accumulation of extracellular toxic peptides, such as Aβ, that in turn act to reduce synapse number.^8,10 Consistent with this idea, brain regions with the highest basal activity are the most affected by Aβ plaque load.¹¹ However, other lines of evidence suggest that activity might have a protective role, as shown by experiments that raised mouse model of neurodegeneration in “environmentally enriched” conditions. Indeed animals raised in enriched environments show a delay in onset/progression of neurodegeneration.^12,13 Moreover, epidemiological studies suggest that educational level is negatively correlated with likelihood of Alzheimer disease. Thus the role of activity in synaptic degeneration remains disputed.

To establish a causal role for activity in the control of synaptic loss, tools are needed that allow us to interfere with activity during the whole period of degeneration. Botulinum neurotoxin type A (BoNT/A) has been used as a tool to block synaptic transmission in the PNS for many years^14,15 and its mechanism of action is well understood. By cleaving SNAP-25, the neurotoxin prevents the formation of a functional SNARE complex and thus inhibits fusion of the synaptic vesicles with the plasma membrane.^14,15 The blocking effect of BoNT/A, particularly on glutamatergic synapses, has been clearly demonstrated after direct exposure of the toxin to central neurons.^16-19 We therefore exploited this neurotoxin to test whether synaptic degeneration in the ME7 prion disease model is activity dependent.¹

We found that infusion of BoNT/A into the hippocampus of naïve animals led to hypertrophy of the presynaptic terminal with an accumulation of synaptic vesicles and despite the lack of formation of normal SNARE complexes there was only a about 5% of synaptic boutons undergoing degeneration.¹ In independent experiments, injection of BoNT/A into the hippocampus was performed at 4 weeks post-inoculation with the ME7 prion agent prior to the onset on detectable synapse degeneration. At 12 weeks into prion disease progression, synapse degeneration is conspicuous in the stratum radiatum of the hippocampus (see Figure 1) and at this time point the extent of synapse degeneration was not different between prion animals with or without BoNT/A. The morphology of the degenerating synapses was also indistinguishable between the two groups.¹

The lack of impact of BoNT/A on synaptic degeneration in the ME7 prion animals allows us to rule out both synaptic activity and synaptic vesicle recycling as a precipitating/contributing factor in the degeneration of the presynaptic element. We initially hypothesized that the toxic misfolded prion protein, still ill-defined at the present time,²⁰ might be taken up by endocytosis during synaptic vesicle recycling and lead to demise of the presynaptic terminal (Fig. 2A). Synaptic vesicle exocytosis could also be conceived as a means by which the toxic prion agent is extruded from the cell and interacts with membrane-bound naïve prion protein (PrP^c), thus leading to further generation of toxic misfolded protein (Fig. 2B). If these concepts were correct, then blockade of synaptic transmission should slow down synapse degeneration, by decreasing entry and/or further generation of misfolded prion protein. This is in contrast to our experimental findings,¹ which led us to conclude that critical steps in the induction of synapse degeneration by the prion agent proceed independent of synaptic vesicle recycling (Fig. 2C).

Figure 2. Schematics to illustrate how misfolded prion protein might lead to the degeneration of a typical presynaptic terminal (top). In (A), the misfolded prion protein is taken up as a Trojan horse during synaptic vesicle recycling and damages the synaptic vesicles leading to degeneration of the vesicles and subsequently the terminal. Degenerating vesicles are indicated by a dotted membrane. In (B) it is postulated that the misfolded prion protein is released during exocytosis and then binds surface, naive prion protein (PrP^c) triggering its conversion to a toxic form that is recycled back into the synaptic vesicles, thus leading to vesicle degeneration over time. One or both of these pathways may be involved and blocking synaptic vesicle recycling with BoNT/A would be expected to delay or preserve the synaptic terminal. In the absence of an effect of BoNT/A, we propose that the misfolded protein enters the terminal via a synaptic vesicle-independent route (C); note blockade of vesicle fusion with the plasmalemma) and exerts its toxic action either at the plasma membrane or within the terminal.

If uptake of a species of misfolded protein is important this still may happen via other routes including the binding to cell surface PrP^c which then undergoes endocytosis and intracellular trafficking via routes independent of the synaptic vesicle cycle. The toxic peptides might also directly bind to the phospholipid bilayer and perturb its fine structure,²¹ resulting in signaling changes and toxic effects proceeding via pathways largely independent of electrical activity. Recent data suggest that the misfolded prion protein may evoke a sustained unfolded protein response that leads to the inhibition of protein translation via phosphorylation of the α subunit of eukaryotic translation initiation factor (eIF2alpha), with subsequent synapse degeneration and neuronal loss.²² At the present time it is not clear whether the shutdown of protein translation takes place at the cell soma or at the synapse.

It has been suggested that SNARE complex assembly, its turnover and efficient degradation is an important component of synaptic integrity in neurodegenerative conditions.^23,24 CSP-α is a chaperone suggested to play an important role in maintaining the integrity of the SNARE protein complex interaction during their rapid recycling in synaptic activity and knockout of this protein leads to a synapse degeneration phenotype.^25,26 Recent data show that the CSP-α −/− neurodegeneration phenotype is exacerbated by a reduction in the expression of SNAP-25, but can be largely reversed by overexpression of SNAP-25.²³ On the other hand, the neurodegeneration of the CSP-α −/− mice is, perhaps surprisingly, not affected by the overexpression of a non-functional BoNT/A-cleaved SNAP-25,²³ which in contrast to the overexpression of SNAP-25 would be expected to inactivate synaptic transmission. The inference is that activity is protective against synaptic degeneration in CSP-α −/− mice. Sharma et al.²³ have suggested that a reduction of SNAP25 exacerbates CSP-α −/− phenotype by the generation of excess levels of syntaxin-1 and synaptobrevin -2, which are no longer complexed to SNAP-25 and these reactive molecules might engage in inappropriate protein interactions that lead to synapse degeneration. However, it is important to note that the BoNT/A-truncated form of SNAP-25 is still able to bind syntaxin-1 on the plasma membrane, even if this interaction is not permissive for synaptic vesicle release.²⁷ It has been suggested that dysfunctional SNARE-complex assembly could contribute to neurodegeneration in Alzheimer’s and Parkinson’s disease.²⁴ An analysis of postmortem brain tissue from Alzheimer and Parkinson disease patients found evidence of reduced SNARE-complex assembly in the brain tissue samples.²⁴ However, in the progression of prion disease there is little evidence of disruption of the SNARE complexes in the hippocampus despite the significant synapse degeneration.²⁸

We should perhaps be wary of drawing too close an analogy between the genetic synaptic loss found in CSP-α −/− mice and the degeneration elicited by the presence of a misfolded protein. In the CSP-α −/− mice, the phenotype can be rescued by the overexpression of α-synuclein,²⁹ another synaptic vesicle associated protein. Burrè et al.³⁰ have shown in vitro that α-synuclein binds the SNARE complex leading to the suggestion that α-synuclein plays a role in determining SNARE complex levels in an activity dependent fashion. One might thus predict that the loss of α-synuclein would exacerbate synapse degeneration in the ME7 model but comparison of a strain of mice lacking α-synuclein with a strain expressing normal levels revealed no difference in the extent of synapse degeneration in ME7 mice.³¹

Conclusion

The loss or degeneration of synaptic contacts plays a major role in the progression of neurodegenerative disease but we know remarkably little about the molecular events that underlie this process. Our studies in murine prion disease strongly support the idea that the degeneration of the presynaptic compartment occurs independent of synaptic activity and synaptic vesicle recycling. New approaches are needed to understand how the misfolded protein triggers this devastating disconnection in the brain.

Caleo M, Restani L, Vannini E, Siskova Z, Al-Malki H, Morgan R, et al. The role of activity in synaptic degeneration in a protein misfolding disease, prion disease. PLoS One. 2012;7:e41182. doi: 10.1371/journal.pone.0041182.