There are important diseases of the CNS that are associated with the accumulation of misfolded proteins either extracellularly, such as Alzheimer's disease and prion diseases, or intracellularly, such as Parkinson's disease and ALS (amyotrophic lateral sclerosis) (Soto, 2003
). A significant risk factor for many of these chronic neurodegenerative diseases is old age and with increasing life expectancy their prevalence is increasing. A major focus of research has been to understand the processes that link the presence of a misfolded protein and the degeneration of neurons, with the ultimate aim of preventing the neuronal degeneration. At present, this linkage is poorly understood (Aguzzi et al., 2008
; Soto, 2003
). Although there is evidence that an early and significant part of the pathology of these diseases is the dysfunction, degeneration or loss of synapses (Conforti et al., 2007
; Saxena and Caroni, 2007
), the demonstration that a particular synapse in the CNS becomes dysfunctional or degenerates prior to either changes in or the demise of the cell body of origin is not a simple matter; this is true of neuron populations in both the human brain and in experimental models. In Alzheimer's disease, synaptic degeneration is an important correlate of the degree of cognitive impairment (Scheff and Price, 2006
) and the loss of dendritic spines from neurons is well documented. We do not know, however, precisely where synaptic degeneration was initiated and whether there are changes in the cell body or the axonal transport of essential synaptic components prior to synaptic changes. The investigation of these early components of neurodegeneration, be they in the pre- or post-synaptic elements, is greatly aided by the development of animal models that simulate at least some components of the human neurodegenerative disease. The synaptic loss that has been documented in human neuropathology has been demonstrated in a number of these models (). Temporal investigation of these models is often consistent with a linear sequence in which the disease causing insult leads to synaptic dysfunction and loss that precedes eventual neuronal cell loss (), although alternative pathways to neuronal loss may also operate (). A simple progression from synapse dysfunction to degeneration and then cell loss should not, however, be assumed at the present time in all chronic neurodegenerative diseases.
Human pathology and animal models: the case for compartmentalized neuronal degeneration with chronic neurodegeneration following a sequential degeneration process or a simultaneous degeneration of all compartments
Schemes to illustrate the temporal relationship between the presence of an accumulating neurotoxic misfolded protein and the sequence of events leading to neuronal loss in chronic neurodegeneration
A valuable system in which it has been shown that synaptic degeneration precedes the demise of the cell soma is the animal models of ALS. In transgenic animals, overexpression of mutated forms of the enzyme SOD-1 (superoxide dismutase-1) leads to the death of spinal motor neurons (Turner and Talbot, 2008
). In these mice, the peripheral synapses in a defined muscle with their cell bodies of origin in the appropriate spinal cord segment are readily studied and it has been shown that synapses at the neuromuscular junction degenerate prior to the cell soma (Frey et al., 2000
; Fischer et al., 2004
). However, an elegant analysis of retrogradely labelled motorneurons demonstrates up-regulation of the ER (endoplasmic reticulum) stress pathway prior to the loss of neuromuscular junction synapses and eventual loss of the cell body (Saxena et al., 2009
). Although these biochemical changes at the cell body are the first outward sign of the impending demise of the synapse and cell body, microglia activation precedes even these changes. This paper highlights the importance of knowing the neuronal cell body of origin of synapses, which makes it possible to demonstrate cell body-related changes that may precede both degeneration of the synapse and morphological changes in the cell body. Importantly, it remains to be shown whether ER stress proteins in the neuromuscular junction synapses have also been up-regulated and, if they are, whether changes in expression at the synapse precede, follow or coincide with changes in the cell body.
In the case of protein misfolding, diseases dominated by the deposition of extracellular aggregates the evidence suggests a similar sequence of synapse dysfunction then synapse degeneration followed by neuronal loss. In mouse models of Alzheimer's disease that overexpress hAPP (human amyloid precursor protein) with pro-amyloidogenic point mutations, or in combination with other genes related to familial Alzheimer's disease such as presenilin-1, there are widespread deposits of amyloid in the brain. Many of these models show synaptic loss (), but limited evidence of neuronal loss, indicating that synaptic degeneration can precede the loss of the cell soma. The synaptic loss is associated with both electrophysiological deficits such as a reduction in LTP and changes in cognitive behaviour ().
Our own experience has involved prion disease in mice. In these studies, we initiate degeneration by selectively introducing the prion agent into the hippocampus and analyse the evolution of disease pathology associated with the accumulation of misfolded prion protein. The loss of synapses has been reported in mouse models of prion disease prior to the onset of neuronal loss. Electron microscopy studies show that as many as 25% of the synapses in the stratum radiatum are lost at a time when there is no detectable neuronal loss from CA3, the neurons of origin of the vast majority of these glutamatergic synapses (Jeffrey et al., 2000
; Siskova et al., 2009
). The loss of synapses is proposed as the substrate of behavioural deficits at early stages of disease (Cunningham et al., 2003
), but it is unclear whether the defects in either behaviour or LTP (Chiti et al., 2006
) can be accounted for by synaptic dysfunction prior to synaptic degeneration.
Morphology of synaptic degeneration
Although there are numerous reports documenting synaptic loss in particular brain regions during chronic neurodegenerative disease, what we know about the degeneration process per se
and the events that initiate it is much more limited. A significant change in thinking about synaptic degeneration has come about from the discovery of the Wlds
mouse (Lunn et al., 1989
; Mack et al., 2001
). Transection of an axon in either the PNS (peripheral nervous system) or CNS leads to the rapid degeneration of the distal synapses and the axon, Wallerian degeneration. In the Wlds
mouse Wallerian degeneration of synapses and axons in both the PNS and CNS is dramatically delayed although, as in wild-type mice, CNS Wallerian degeneration and loss of the ability of axons to conduct action potentials is slower than in the PNS (Lunn et al., 1989
; Perry et al., 1991
). Prior to the discovery of this mouse, there was little reason to believe that the degeneration of the axon and synapses was anything other than the passive degeneration of processes isolated from the cell soma, their source of support. The discovery that the chimaeric Wlds
protein, which arose as a spontaneous mutation in a substrain of C57BL mice, leads to dramatic slowing of Wallerian degeneration (Mack et al., 2001
), implies that in wild-type animals there must be biochemical processes to both activate and inhibit the axon and synapse degeneration pathways. The current concept of a neuron with distinct compartmentalized degeneration has been reviewed elsewhere (Gillingwater and Ribchester, 2001
) and the molecular basis of the mode of action of Wlds
is being hotly pursued (Coleman and Freeman, 2010
). If synapse degeneration is an active process, it is critical to understand the sequence of events involved.
In the PNS, when synapses at the neuromuscular junction undergo Wallerian degeneration, there is a withdrawal of the presynaptic ending from the postsynaptic sites, with preservation of both the presynaptic membrane and the synaptic vesicles (Winlow and Usherwood, 1975
). Detailed analysis of the degeneration process is facilitated in Wlds
mice with slowed degeneration, and electron microscopy studies show that withdrawal of the presynaptic terminal is associated with retention of synaptic vesicle content, invasion of the terminal by neurofilaments and envelopment of terminal fragments by Schwann cells (Gillingwater et al., 2003
). The retraction of the synapse during PNS Wallerian degeneration has clear parallels with modelling of the neuromuscular junction synapse in development. The retracting synapses shed small organelle-rich membrane-bound portions of the axon, so-called axosomes, which are engulfed by Schwann cells (Bishop et al., 2004
). Lysosomes in the withdrawing axon and the Schwann cell have been described and are likely involved in degrading the terminal (Song et al., 2008
In contrast to the PNS, during Wallerian degeneration in the CNS, degenerating synapses do not withdraw from the PSD (postsynaptic density). The presynaptic terminal has electron dense cytoplasm with the apparent loss of integrity of synaptic vesicles and other organelles, but the presynaptic membrane remains intact and closely adhering to the PSD membrane (Lund and Lund, 1971
; Nadler et al., 1980
). Studies of Wallerian degeneration in the corticostriatal pathway in both wild-type and Wlds
mice show that the morphology of the degeneration process is identical, although delayed, in the Wlds
mice (Gillingwater et al., 2006
). In a small number of instances, the portion of the axon immediately pre-terminal to the degenerating synapse was morphologically intact, consistent with the idea that degeneration of the synapse is indeed a compartment-specific event.
It is clear from the available data that degeneration of CNS synapses induced by an acute injury is distinct from that seen in the PNS at the neuromuscular junction. During acute degeneration of synaptic terminals in the PNS there is involvement of the Schwann cells that phagocytose some of the degenerating material, while in the CNS there is little evidence of direct involvement of either the microglia or astrocytes. Despite their lack of involvement in synapse phagocytosis, microglia rapidly respond to the presence of synaptic degeneration with increased or de novo
expression of different proteins and a change in morphology (Rao and Lund, 1993
; Jensen et al., 1999
). The nature of signals from degenerating synapses that lead to activation of the microglia are not known although there are many potential candidates including neurotransmitters and other low-molecular mass mediators (Hanisch and Kettenmann, 2007
). While the activation of microglia and the subsequent degeneration of synapses are coincident in time and space, the association may not be a causative one.
Synaptic degeneration in chronic neurodegeneration
In chronic neurodegenerative diseases in humans, the study of synapse degeneration is limited by the availability of tissue with the appropriate tissue fixation and preservation. Animal models of protein misfolding disease offer an opportunity to study these events, in particular, in circuits where the cell body of origin is well defined. Prion disease in mice is a highly tractable laboratory model of chronic neurodegeneration caused by the presence of a misfolded protein (Aguzzi et al., 2008
). Unlike many other models, the precise timing and locus of the site of initiation of disease is under the control of the experimenter. The appearance of the first behavioural deficit in the ME7 prion model appears in hippocampal-dependent tasks (Guenther et al., 2001
) and, since the circuitry of the hippocampus is well known, this offers the opportunity to dissect the anatomical and electrophysiological substrate of these behavioural deficits. At the time of appearance of the first behavioural deficit there is no loss of neuronal cell bodies, but there is significant loss of synapses from the stratum radiatum of the hippocampus, as detected by synaptophysin immunocytochemistry (Cunningham et al., 2003
). The axons of the CA3 pyramidal cells form en passant synapses on the CA1 pyramidal cells and the varicosities, presumed synapses, are aligned like a string of beads along the axon. The cell bodies of origin of these synapses, the CA3 pyramidal cells, do not degenerate during the course of the disease, but during late stages of the disease they shrink and show abnormal vacuolation of their dendrites (Belichenko et al., 2000
; Gray et al., 2009
). Quantitative electron microscopy studies have confirmed the loss of the synapses (Jeffrey et al., 2000
; Siskova et al., 2009
) and allow a study of the morphological events.
The degenerating synapses are recognized by the presence of an electron dense cytoplasm, and the loss of definition of the vesicle integrity () (Siskova et al., 2009
). The appearance of the presynaptic element is very similar to that seen in Wallerian degeneration but with a notable difference. In the animals with prion disease, the presynaptic membrane not only remains intact in close apposition with the PSD, but now the PSD also appears progressively curved around the degenerating presynaptic element () and, in advanced stages, it appears that the PSD has almost completely enveloped the degenerating terminal (). The progressive darkening of the presynaptic terminal has also been reported in hAPP transgenic mice and there is a notable increase in dark organelles that may be either lysosomes or autophagic vacuoles (Adalbert et al., 2009
). This is in contrast to the presynaptic endings seen in prion disease and also in Wallerian degeneration, where there is no apparent increase in lysosomes or autophagic vacuoles. We should also be cautious about drawing too close a parallel between synapse degeneration in Wallerian degeneration and degeneration mediated by accumulation of an extracellular misfolded protein. Wlds
mice do not show prolonged survival when challenged with prion disease (Gultner et al., 2009
; VH Perry, H Scott and D Boche, unpublished results).
Perhaps the most striking feature of the prion-induced synaptic pathology is the increased curvature of the PSD. This is a remarkable morphological change that requires profound remodelling of the proteinaceous cytomatrix structure that acts as an organizer of postsynaptic signalling and is the core of the transynaptic process that ensures the tight junction-like association of the pre- and post-synaptic specialization. It is clear that the PSD structure is susceptible to changes in the biochemical composition that impact on plasticity. The idea that the pathological plasticity represents a biologically relevant pathway is reinforced by observations highlighting similar morphological changes in PSDs in hippocampal synapses following the induction of LTP (Connor et al., 2006
) and also during reactive synaptogenesis (Marrone et al., 2004
). It is suggested that the change in curvature is a compensatory mechanism by which the probability of transmitter release is increased to improve synaptic efficacy (see Marrone et al., 2004
The presence of degenerating synapses enveloped by the dendritic spine raises a number of interesting questions such as what happens to the dendritic spine and to the axon. We have not yet addressed in the prion disease model whether the degenerating bouton or some of this material is internalized into the spine or dendrite cytoplasm but there are precedents for the envelopment of large structures by neighbouring cells in both pathology and development. Most remarkable is the process of entosis in which whole cells, likely cancerous in nature, are engulfed by neighbouring cells (Overholtzer et al., 2007
). In the nervous system, the envelopment of synapses by the neuronal cell body both in normal development and following axon injury has been described (Borke, 1982
; Ronnevi, 1979
) as has phagocytosis of diverse materials by neurons (Bowen et al., 2007
). In prion disease, there is a loss of dendritic spines from hippocampal pyramidal cells, but the relationship with the presynaptic changes is not established (Belichenko et al., 2000
). In vivo
imaging studies of cortical pyramidal cells during prion disease progression reveal that dendritic spines are retracted over a period of several days, with the appearance of dendritic varicosities (Fuhrmann et al., 2007
). A further intriguing question is whether the degeneration of a synapse along the Schaffer axon leads to the loss of only that synapse or to the degeneration of the axon distal to the degenerating synapse as well. Does the spine envelopment compromise the survival of the distal axon? Studies are in progress to try and resolve this matter.
Microglia in synaptic degeneration
The electron microscopy studies allow us to address the issue of whether microglia are involved in the phagocytosis of degenerating synapses. In the prion model, there is no evidence at any stage during the envelopment of the synaptic terminal by the PSD that processes of microglia, or indeed other glia, are directly involved in the degeneration process (). Three-dimensional reconstructions from both conventional and dual-beam electron microscopy rule out the presence of non-neuronal processes between the presynaptic and postsynaptic elements (Siskova et al., 2009
). In other models of either Wallerian degeneration in the CNS or chronic degeneration, the synaptic boutons do not appear to be enveloped by microglia processes (Adalbert et al., 2009
; Scott et al., 2010
). The microglia respond early to the presence of prion pathology in the hippocampus (Betmouni et al., 1996
) and although the microglia are able to phagocytose latex beads delivered to the hippocampus of prion-diseased animals (Hughes et al., 2010
) they do not engulf the degenerating synapses.
The microglia in the hippocampus of mice with prion disease have an activated morphology but are associated with an anti-inflammatory phenotype, dominated by the presence of transforming growth factor-beta and prostaglandin-E2
, akin to that seen in macrophages that have phagocytosed apoptotic cells (Perry et al., 2002
; Savill et al., 2002
). Given the suggestion that synapse degeneration is an active compartmentalized auto-destructive process (Conforti et al., 2007
; Gillingwater and Ribchester, 2001
) one hypothesis would be that this phenotype arises as a consequence of phagocytosis of the degenerating synapses. This is clearly incorrect, since the microglia do not seem to be involved. However, at this stage, we cannot rule out the possibility that the conspicuous shrinkage of the presynaptic element is associated with the shedding of exosomes, nanoparticle-sized membrane vesicles, which has been described in the PNS (Bishop et al., 2004
) and also for CNS neurons in culture (Faure et al., 2006
). It has been suggested that supernumerary synapses in development and synapses in neurodegenerative conditions might be opsonized by complement prior to phagocytosis by microglia (Stevens et al., 2007
). It is unclear whether the complement cascade is activated or decorates the degenerating synapses in the early stages of the evolution of prion disease. The possibility that the slow accumulation of misfolded protease-resistant prion protein [PrPSc
(abnormal disease-specific conformation of PrP)] activates the microglia is difficult to establish in vivo
, but it is notable that in peripheral tissues, such as the spleen where PrPSc
is also deposited, the local macrophages do not show an activated phenotype (Cunningham et al., 2005
). The factors that lead to the morphological activation of the microglia with an anti-inflammatory phenotype are yet to be identified.
Biochemical events in synaptic degeneration
In addition to morphological studies of synapse degeneration, some attempts have been made to investigate the biochemical events associated with the morphological changes. Although a number of studies have shown the loss of synaptic proteins in late-stage disease in both human and animal models, these are not particularly informative, since the loss of synaptic proteins is unsurprising if there is also neuronal degeneration. We have attempted to address this issue by studying the time points in prion disease when there is ongoing synaptic loss prior to detectable neuronal loss. The quantification of the synaptic proteins from an isolated region of the diseased brain is not straightforward, since it is not immediately clear what a particular synaptic protein should be quantified relative to. The usual housekeeping proteins used in many studies are of little intrinsic value in a condition where the non-neuronal cells are dramatically changing in number and changing their proteome (see Gray et al., 2009
for discussion). We have thus used a method to quantify the absolute protein loading on Western blots and then studied both pre- and post-synaptic protein levels at different stages of disease evolution. A number of proteins associated with the synaptic vesicle membrane were the first to show reduced levels of expression, including VAMP-2 (vesicle-associated membrane protein-2), synaptophysin and the chaperone CSP (cysteine string protein) (Gray et al., 2009
), and these proteins were also reduced when compared with the levels of PSD-related proteins. The loss of presynaptic proteins is consistent with the early morphological changes in which there is a loss of vesicle integrity but an apparently morphologically intact PSD.
The changes in CSP expression are of particular interest, as it has been shown that deletion of the CSP gene in mice (CSP−/−
mice) leads to a synapse degeneration phenotype and dramatically shortened lifespan of the mice (Fernandez-Chacon et al., 2004
). Whether microglia or astrocytes are involved in any stage of synaptic degeneration in the CSP−/−
mice is not known. The degeneration phenotype of CSP−/−
mice can be rescued by overexpression of the synaptic protein α-synuclein (Chandra et al., 2005
), suggesting an important interaction between these two proteins. Hence, one might expect that the deletion of this gene would lead to accelerated synaptic degeneration in chronic neurodegeneration. A comparison of prion disease progression by behavioural, biochemical and anatomical methods surprisingly revealed no difference in disease progression in mice with or without α-synuclein (Asuni et al., 2010
). A number of in vitro
models have interesting parallels with the loss of presynaptic proteins and the changes in LTP described in the prion model (Chiti et al., 2006
). Parodi and colleagues (Parodi et al., 2010
) have shown that chronic treatment of neurons with Aβ oligomers leads to the loss of presynaptic proteins and reduced spontaneous activity. In another model, overexpression of human α-synuclein in hippocampal neurons has a profound effect in down-regulating the expression of a number of important proteins in synaptic vesicles including VAMP-2, piccolo and synpasin-1 (Scott et al., 2010
). The authors report abnormally large synaptic vesicles, suggesting a fusion of vesicle membranes.
At the neuromuscular junction, the Wallerian degeneration of synapses is not associated with a loss of synaptic vesicles but there may be a loss of integrity of mitochondria (Gillingwater et al., 2003
). The idea that oxidative stress contributes to chronic neurodegenerative disease is widespread, but how it might specifically contribute to degeneration of the synaptic compartment has not been addressed. We have investigated whether mitochondria in the hippocampus of prion-diseased mice with synaptic loss are also affected. We found that although the mitochondria density remains normal, the mitochondria show subtle morphological abnormalities and a reduction in cytochrome c
oxidase activity (Siskova et al., 2010
). The change in mitochondrial function is consistent with magnetic resonance spectroscopy measurements showing a reduction of the NAA (N-acetylaspartate)/choline and NAA/creatine ratio in the hippocampus of prion-diseased mice at the time of synaptic loss (Broom et al., 2007
). NAA is widely accepted as a marker of neuronal functional integrity and is closely linked with a bio-energetic role in neuronal mitochondria (Moffett et al., 2007
). It is at present not clear whether the mitochondrial changes are a cause or a consequence of the synaptic degeneration and the events leading to mitochondrial cytochrome c
oxidase impairment are not known. However, during the period of early synaptic loss and mitochondrial abnormalities, there is an enhanced expression and activity of nNOS in the stratum radiatum (Picanco-Diniz et al., 2004
). This could, as discussed earlier, modulate synaptic function and additionally impair mitochondrial function.