The results presented in this study demonstrate a CaN-dependent mechanism of dendritic spine loss in the pilocarpine model of SE. A dramatic increase in CaN activity and concentration occurs in the crude SPM fraction of hippocampal and cortical tissues at or near the onset of continuous seizure activity. Coupled with our previous histochemical results (Kurz et al., 2003), this demonstrates an increase in the effective amount of CaN phosphatase activity in dendritic regions of neurons. This study elucidated one pathological consequence of this increased postsynaptic CaN activity by examining CaN's effects on dendritic actin stability. First, we have shown a CaN-dependent dephosphorylation of the actin-depolymerizing factor, cofilin. Like the increase in CaN concentration and activity, this dephosphorylation develops shortly after the onset of continuous seizure activity. The expected biochemical consequences of an SE-induced activation of cofilin were also found to occur in a CaN-regulated manner, with a SE-dependent increase in cofilin–actin binding and a subsequent SE-induced depolymerization of dendritic actin. Finally, the CaN-dependent depolymerization of dendritic actin led to SE-induced dendritic spine loss, which was demonstrated histologically in several brain regions and was blocked with the CaN inhibitor FK506. These findings demonstrate a cellular pathway, through which SE induces dendritic spine loss.
SE and spine loss
The observed SE-induced reduction in dendritic spine density may be part of the long-term neurological pathology that is associated with SE. As the primary site of excitatory neuronal synapses, and given their importance in models of learning and memory, spines appear to be critical for normal cognition. Studies in animal models have documented cognitive difficulties associated with SE, both in adult and developing animals (Rice et al., 1998). While SE-induced neuronal death undoubtedly accounts for some of this loss of cognitive function, other, more subtle, mechanisms—such as spine loss—are likely responsible as well. Depending on the duration of the SE-induced decrease in dendritic spine density, the mechanism described in this study could be a mechanism underlying SE-induced cognitive dysfunction. In one previous study, dendritic spine density rebounded in the weeks following SE, although with significant alterations in spine morphology and location. Spine density then decreased again once chronic seizure activity ensued (spontaneous recurrent seizures are a common consequence of the pilocarpine model of SE) (Isokawa, 1998). These findings tend to argue against the acute spine loss described in this study as the cause of long-term cognitive dysfunction, as it does not persist long enough to account for chronic deficits of cognition. However, the long-term decrease in spine density seen in many models of recurrent seizures may involve a chronic, lower-intensity activation of the CaN-regulated mechanism described above. This persistent decrease in spine density could be involved in chronic deficits of cognitive function. More study is needed to determine the role of CaN in chronic epileptic states. On the other hand, the acute, CaN-regulated spine loss described above may be involved in another SE-associated pathology, epileptogenesis.
In the pilocarpine model of SE, spontaneous recurrent seizures typically begin approximately 2 weeks after the prolonged seizure episode (Turski et al., 1989). No epileptic activity is seen during this quiescent period, but there is a great deal happening on a cellular level. It is thought that the biochemical and structural changes that lead to recurrent seizure activity are being completed during this time. While the exact mechanisms responsible for epileptogenesis remain the subject of intense study and debate, dendritic spine plasticity certainly is a promising candidate for this role. The loss and subsequent regrowth of dendritic spines could represent a pathological reorganization of synaptic networks leading to the formation of epileptic foci in the brain. It is especially interesting that spine loss has been documented in the dentate gyrus of the hippocampus. Network reorganization and neuronal hyperexcitability in this region are frequently proposed as possible mechanisms of epileptogenesis (Sloviter, 1999; Bragin et al., 2000). Furthermore, after the SE-associated loss of spines, it has been shown that their regrowth in this region is colocalized with sites of mossy fiber sprouting (Isokawa, 2000). Thus, an outgrowth of axonal structures that has been previously implicated in epileptogenesis seems to coincide with a reorganization of postsynaptic structures, all of which is occurring in a region that seems to be critical in the development of temporal lobe epilepsy. Considered in the context of epileptogenesis, the CaN-mediated mechanism of spine loss presented in this paper may help to explain the results of several other studies. FK506 ameliorated spine loss in the present study, while other researchers have documented that CaN inhibitors can prevent epileptogenesis in both a kainic acid model of SE (Moriwaki et al., 1998) and a kindling model of epilepsy (Moia et al., 1994; Moriwaki et al., 1996). Further research may determine if these two effects are related.
Cellular mechanism of spine plasticity
Spine plasticity has long been observed in both chronic epilepsy and acute SE models (Wong, 2005), although the mechanisms that underlie this spine loss have not yet been determined. A great deal of research suggests that CaN modulates neuronal spine density and morphology under normal conditions, with the enzyme having been previously shown to cause calcium-stimulated spine plasticity in several nonpathological model systems. Halpain et al. demonstrated a loss of spines in cultured hippocampal neurons in response to NMDA application. CaN was shown to colocalize with F-actin at synapses, and CaN inhibitors blocked NMDA-mediated spine loss in these neurons, strongly suggesting a CaN-mediated spine loss (Halpain et al., 1998). Similarly, Zhou et al. described an long-term depression (LTD)-associated spine shrinkage that required both NMDA and CaN to occur (Zhou et al., 2004). Under physiological conditions, it is highly probable that CaN-mediated spine plasticity is part of a learning and memory mechanism, considering the importance of both CaN and spines to learning models. However, with the profound increase in CaN levels at the synapse that we have observed in SE, this regulation of spine plasticity may become pathologically active, resulting in a detrimental loss, and then reorganization, of synaptic contacts.
The present study documented a profound increase in CaN concentration and activity in the crude SPM fraction of neuronal tissue of animals that had undergone as little as 5–10 min of continuous seizure activity. As we have described previously, continuous seizure activity typically began approximately 10 min after the first discrete seizure in this model (Singleton et al., 2005b). No detectable increase was seen, however, in animals that experienced discrete, but not continuous, seizure activity. There are two possible explanations for this. The onset of SE may be essential for significant changes in CaN to occur, or these discrete seizures may simply not have been of sufficient duration to induce a measurable translocation of the enzyme (although a longer, but still isolated, seizure may prove sufficient). Regardless, CaN translocation seems to be a neuronal reaction to prolonged seizure activity. Such a translocation of CaN to the synapse may initially be a physiological defense mechanism for the neuron in the face of excessive excitation, as CaN is known to negatively modulate neurotransmission through the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and NMDA subtypes of glutamate receptor. Perhaps such a mechanism, occurring near the onset of continuous seizures, represents a pathway by which seizure activity can be terminated in brains that are not prone to SE. However, as SE progresses and worsens, this sustained increase in synaptic CaN activity also could play a pathological role in neurons. A number of recent studies support such a hypothesis. For example, it has long been known that administration of CaN inhibitors prior to SE prevents chronic SE-related behavioral pathologies, such as a SE-induced loss of cognitive abilities and the development of spontaneous continuous seizures (Moriwaki et al., 1998). Several mechanisms have been proposed as pathological roles for CaN in seizure disorders, including negative modulation of the GABA receptor (Sanchez et al., 2005; McNamara et al., 2006) and—the focus of the current study—modulation of dendritic spines.
CaN may perform this pathological modulation of spine morphology via its regulation of the actin-depolymerizing factor, cofilin. Cofilin is a small peptide that, when dephosphorylated, binds to F-actin and causes its depolymerization (Agnew et al., 1995; Bamburg, 1999). Cofilin has been shown to regulate the structure of dendritic spines by inducing the depolymerization of their actin cytoskeleton (Meng et al., 2004; Sarmiere & Bamburg, 2004; Zhou et al., 2004). Recent studies have shown that CaN induces cofilin dephosphorylation (and thus subsequent actin depolymerization) either directly (Meberg et al., 1998) or more likely, indirectly via an intermediary phosphatase known as slingshot (Wang et al., 2005), thus making a mechanism involving CaN activation of cofilin and subsequent spine loss quite plausible. In fact, one recent study has already shown spine regulation under some physiological conditions through a mechanism dependent on both CaN and cofilin (Zhou et al., 2004).
The data described in this study clearly indicate a CaN-dependent regulation of cofilin in SE. First, the SE-induced dephosphorylation of cofilin required the onset of continuous seizure activity, a requirement shared with the increase in SPM CaN and SE-induced dendritic spine loss. In fact, the timing of this loss of SPM cofilin phosphorylation coincides perfectly with the increase in SPM CaN concentration and activity. Furthermore, both of the CaN inhibitors cyclosporin A and FK506 blocked SE-induced cofilin dephosphorylation. The fact that two different CaN inhibitors blocked SE-induced cofilin dephosphorylation strongly argues in favor of a CaN-dependent mechanism, although some modest SE-induced dephosphorylation did occur in the presence of effective doses of each inhibitor. This is not in itself surprising, as a number of other mechanisms exist for the regulation of cofilin phosphorylation. Activation of these pathways in SE is certainly possible, and merits future study. However, CaN-mediated dephosphorylation represented a major portion of the observed decrease in phosphocofilin immunoreactivity. This CaN-dependent dephosphorylation of cofilin did indeed activate the molecule, as SE was also shown to lead to a CaN-dependent increase in cofilin–actin binding and a CaN-dependent depolymerization of actin. While the observed change in the F/G actin ratio was relatively modest, when one considers the prevalence of actin in all cell types and cellular structures in the brain, even a 20% difference in the relative amounts of F- and G- actually represents a quite profound structural change. This structural change manifested itself in a CaN-dependent loss of dendritic spines. As other researchers have previously, we observed a SE-dependent loss of dendritic spines after 1 h of SE. Considering the timing of the CaN-induced cofilin dephosphorylation, this loss of spines may, in fact, have occurred even earlier in SE, and future studies should certainly explore synaptic changes at these earlier time points.
The novel findings presented in this study describe a cellular mechanism for actin depolymerization and dendritic spine loss in the pilocarpine model of SE. This spine loss is widespread throughout the forebrain after SE, and is CaN-dependent, involving the actin depolymerizing factor, cofilin. Further research in this area may help elucidate the role of dendritic spine plasticity in SE-associated neuronal pathologies, potentially provide insight into some of the mechanisms underlying epileptogenesis, and provide the basis for future treatment options.