BACKGROUND
excitotoxicity is a neurodegenerative process believed to be the central mediator of brain damage in acute conditions such as brain ischemia and traumatic injury, and an important contributor to a range of chronic neurodegenerative diseases  <cit> .
in excitotoxicity, the malfunction of glutamate  transporters   <cit>  causes accumulation of glu in excitatory synapses and exaggerated stimulation of postsynaptic glu receptors   <cit> .
the excessive influx of ions  into the postsynaptic neurons leads to their cell death via a spectrum of mechanisms that range from necrosis  to apoptosis or even recovery .
despite our familiarity with the first few steps in excitotoxicity, our understanding of the steps following ca2+ influx is very limited.
clinical trials using glurs antagonists ended with disappointment, and recent data suggests that using antagonists to block glur functions might be counterproductive, because glu signaling includes both neurotoxic and pro-survival cascades  <cit> .
these complications emphasize the need to illuminate cell-death-specific signaling cascades in excitotoxicity downstream of glurs.
a considerable number of such excitotoxic mechanisms have been suggested, but in many cases the data that supports a given suggestion is limited to specific excitotoxic paradigms.
one suggested mediator of excitotoxicity implicated in multiple experimental setups is the cam-dependent death associated protein kinase   <cit> .
originally identified by an unbiased screen for mediators of interferon-induced cell death  <cit> , dapk was later recognized as a molecular switch that controls the choice between cell death processes such as apoptosis and autophagy  <cit> .
moreover, an elaborate web of biochemical interactions and functional connections has been revealed, placing dapk in a key position at the center of many critical signaling cascades  <cit> .
a considerable body of evidence suggests that dapk also contributes to cell death in excitotoxicity  <cit> , and its inhibition reduces neuronal loss in models of brain ischemia  <cit> .
several mechanisms have been suggested to explain dapk’s involvement in excitotoxicity, but the issue remains controversial.
one possible mechanism involves dapk’s regulation of autophagy  <cit> , a process that modulates some neurodegenerative conditions  <cit> .
alternatively, an intriguing study suggests the involvement of danger, a protein that contains a region of homology to the nematode protein mab- <dig> and functions as a regulator of the ip3r  <cit> .
indeed danger was found to also interact with and inhibit the activity of dapk  <cit> .
the most cited suggestion in the field attributes the involvement of dapk in excitotoxicity to the potentiation of ca2+ currents through nr2b/glun2b subunit-containing complexes of the nmda- receptor  family of glurs  <cit> .
this suggestion fits well with a proposed leading role for extrasynaptic nr2b/glun2b –containing nmda-rs in excitotoxicity  <cit> .
however, recently the proposed unique significance of nr2b/glun2b –containing extrasynaptic nmda-rs in excitotoxicity has been brought into question  <cit> .
moreover, an earlier study suggests that in some mammalian neurons dapk knockout provides protection from excitotoxicity that is not dependent on nmda-rs  <cit> .
indeed, some cases of excitotoxicity are mediated by another family of glurs, the ca2+ -permeable ampa receptors   <cit> .
these observations suggest that if dapk is widely involved in excitotoxicity, including in cases where nr2b/glun2b is not a main determinant of neurodegeneration, it might act through additional mechanisms to exert its effect.
we hypothesize that many signaling cascades might be involved in specific cases of excitotoxicity, depending on the exact scenario being used to induce it.
however, the key features that constitute the core of the excitotoxic process might be conserved across differences in cell death scenarios and large evolutionary distances, as is the case in apoptosis  <cit>  and autophagy  <cit> .
we therefore set out to study the role of dapk in excitotoxicity in our model of cpar-mediated neurodegeneration in c. elegans  <cit> , where glur-dependent necrosis of central neurons postsynaptic to glu connections is triggered by knockout  of the glut gene glt- <dig>  <cit>  in a sensitized background .
indeed, this model has proven effective in identifying core processes that are conserved between nematode and mammalian excitotoxicity  <cit> .
dapk is particularly well conserved in c. elegans .
the nematode dapk- <dig> is widely expressed , allowing us to test its involvement in nematode excitotoxicity and to study its mechanism of action.
in this study we establish the central role of dapk in glu-triggered neurodegeneration in c. elegans, suggesting that its function is conserved across evolution and excitotoxic scenarios.
we find little or no support for the views that dapk’s regulation of excitotoxicity is mediated through the modulation of synaptic strength, mab- <dig>  or autophagy.
instead, we identify pinn- <dig>  the nematode homolog of the dapk-interaction-partner and phosphorylation-dependent peptidyl-prolyl isomerase pin <dig>  as an important factor in nematode excitotoxicity.
RESULTS
dapk- <dig> has a central role in nematode excitotoxicity
to test the involvement of dapk in nematode excitotoxicity we combined our excitotoxicity strain  with a deletion allele of the nematode homolog of dapk, dapk- <dig>  a gene knockout that was used to confirm the effect of the  nematode dapk in autophagy and innate immunity  <cit> .
in our previously described model of neurodegeneration we use the sensitizing transgenic modification nuis <dig>  where hyperactive gαs and gfp are expressed under the glr- <dig> promoter in ~ <dig> neurons  <cit>  and cause glur-independent stochastic degeneration of ~ <dig> of these at-risk neurons per animal.
when we add the ko of the glut gene glt- <dig> we observe that more of these at-risk neurons degenerate  <cit> .
the glut-ko-triggered exacerbated necrosis in glt-3;nuis <dig> is glur-dependent, and therefore qualifies as nematode excitotoxicity.
nematode excitotoxicity causes neuronal swelling and death that is manifested with characteristic kinetics as gradually and stochastically appearing vacuole-like structures in some of the at-risk postsynaptic neurons.
these vacuole-like structures become more abundant during larval development as the glu signaling system matures , and then decline due to removal of cell corpses by engulfment  <cit> .
we now observe that adding dapk- <dig> ko to this excitotoxicity strain causes a strong and statistically significant suppression of neurodegeneration throughout development .
to further confirm the contribution of dapk- <dig> to nematode excitotoxicity, we overexpressed the wt dapk- <dig> cdna from an extra-chromosomal transgenic construct under a heat-shock promoter  <cit> .
since heat-shock might affect susceptibility to neurodegeneration, we took special care to compare an exact match of treated animals, without or with the dapk- <dig> overexpression transgene.
to that end we took advantage of the fact that the random and partial segregation of the non-integrated overexpression construct allows us to compare transgenic and non-transgenic animals on the same plate exposed to the same conditions.
we observed that dapk- <dig> overexpression resulted in a strong and statistically significant potentiation of necrotic neurodegeneration in postsynaptic neurons in all developmental stages .
together, our data indicate that dapk is an important mediator of excitotoxicity in c. elegans, suggesting that diverse scenarios of excitotoxicity share a common mechanism that assigns a central role to dapk.figure  <dig>
dapk- <dig> is an important mediator of nematode excitotoxicity.
a) dynamics of neurodegeneration in nematode excitotoxicity during development .
dapk- <dig> ko mutation suppresses neurodegeneration in all developmental stages, but does not bring it down to background levels.
b) overexpression of wt dapk- <dig> from a heat-shock promoter enhances neurodegeneration compared to matched controls.
in all bar graphs, error bars represent se.
statistical significance is calculated using z score.
*p <  <dig> ; ***p <  <dig> .
dapk- <dig> ko does not alter presynaptic release or postsynaptic glu response
we used two main guidelines in trying to track the mechanism by which dapk- <dig> regulates excitotoxic neurodegeneration in the nematode: 1) we looked at previous reports suggesting specific mechanisms for dapk’s involvement in mammalian excitotoxicity; 2) we inferred from the general map of dapk’s connectome in other cell processes  <cit>  which additional proteins are plausible candidates for mediating dapk’s effect in nematode excitotoxicity.
one line of evidence suggests that dapk interacts with, and may regulate the function of, syntaxin 1a  <cit> .
syntaxin is part of the general mechanism of vesicular neurotransmitter release, a mechanism that is shared among all neurotransmitters  <cit> .
therefore, an effect of dapk- <dig> ko on any component of the synaptic vesicle release could lead to dapk-mediated changes in excitotoxicity levels.
however, given the ubiquitous expression of this gene, such an effect of dapk on the common synaptic vesicle release mechanism will affect the dynamics of neurotransmitter release in all synapses.
the study of synaptic vesicle release is very well developed in the nematode, and aldicarb is routinely used in c. elegans to identify mutations that cause even modest changes to the general synaptic release mechanism  <cit> .
as aldicarb suppresses the degradation of acetylcholine in the neuro-muscular junction, it causes animal paralysis with a typical dynamics.
mutations that reduce the activity of the general, common vesicle release mechanism  cause a pronounced resistance to aldicarb , while mutations that enhance synaptic release  cause increased sensitivity to aldicarb .
we observed that the sensitivity of dapk- <dig> animals to aldicarb is indistinguishable from that of wt animals, suggesting that dapk- <dig> ko does not modify the synaptic release mechanism, as would have been expected of an effect on syntaxin .figure  <dig> the effect of dapk- <dig> does not correlate with several proposed mechanisms.
a) aldicarb assay measures the overall activity of the common mechanism of synaptic vesicle release.
complexin  mutant is used as example of an oversensitive mutant , while rab- <dig> serves as an example of aldicarb resistant mutant .
dapk- <dig> mutants do not show oversensitivity or resistance, suggesting their level of synaptic release is roughly normal.
b) duration of forward runs during spontaneous mobility is a sensitive reporter of the overall strength of glu synapses that control this parameter.
even small changes in the activity or number of glurs typically modify this behavior.
dapk- <dig> mutants are not different from normal counterparts in either a wt background, in an excessive glu stimulation background , or in the sensitized background  used in our excitotoxicity model.
these three conditions by themselves do not cause excessive command neuron loss, a loss that might otherwise render the assay uninformative as it is suppressible by dapk- <dig>  c) a mutation in the gene encoding the danger-related protein mab- <dig> does not affect nematode excitotoxicity.
d) a mutation in the gene encoding the nematode homolog of camkk does not affect nematode excitotoxicity.
we next asked if dapk modifies the extent of postsynaptic response in the glutamatergic synapses where excitotoxicity occurs .
two behavioral assays have proven very effective in detecting even small changes in the activity level of glutamatergic synapses in c. elegans.
these are the nose touch  assay, and the duration of spontaneous forward mobility assay.
therefore, if dapk- <dig> ko causes changes in the specific glu packaging mechanism in synaptic vesicles , or changes the number or activity-level of glurs in the synapse, this should be reflected in behavioral changes in these assays.
in the particular case of dapk- <dig>  a secondary phenotype of this mutation, namely overgrowth of cuticle on the animal’s nose  <cit> , makes the nose-touch assay less informative.
however, the duration of spontaneous forward mobility depends on the internal balance between forward and backward circuits, and should not be affected by the cuticle aberration.
this assay is very reliable in detecting both under-activity and over-activity of the relevant glu synapses  <cit> , allowing us to use it as a proxy measure for glu synaptic strength.
we did not observe any changes in spontaneous mobility triggered by dapk- <dig> ko, either in wt background or in the background of each of the two components  used in our excitotoxicity model to trigger the intensified glu signaling .
these observations suggest that dapk- <dig> has no strong effect on synaptic release or the overall strength of signaling in glu synapses in c. elegans.
the danger-related protein mab- <dig> and the camkk ckk- <dig> do not exert strong regulation of nematode excitotoxicity
the characterization of dapk as a cam-dependent kinase is particularly intriguing to us, since ca2+ signaling is critical to excitotoxicity in both mammals and nematodes.
we therefore examined dapk-partners that might also be involved in ca2+ signaling.
danger is a mammalian dapk inhibitor that regulates ca2+ release from the er  <cit> .
there is no direct, full-length homolog of danger in the worm genome, but the core of the mammalian protein is thought to be homologous to the nematode protein mab- <dig>  <cit> .
it is therefore reasonable to assume that mab- <dig> will be essential to the role of a hypothetic danger-like complex in c. elegans.
however, a mutation in mab- <dig> had no effect on nematode excitotoxicity .
another important protein that binds mammalian dapk and is involved in ca2+ signaling is camkk.
however, again, a mutation in the only camkk gene in the worm, ckk- <dig>  <cit> , had no effect on excitotoxicity .
these results suggest that these two putative ca2+ signaling regulators and dapk-interaction-partners do not have a large contribution to nematode excitotoxicity.
we also considered the many other proteins that are known to interact with dapk  <cit>  as possible modulators of excitotoxicity, but we could not assign to many of them high priority because either there was no immediate obvious connection to neurodegeneration , there is no clear nematode homolog , the nematode homolog is not known to be active in c. elegans neurons , or the process in which this protein is involved has been shown by us to be not involved in nematode excitotoxicity .
however, dapk is also known to interact with beclin <dig>  a key regulator of the evolutionary conserved process of autophagy, suggesting that autophagy-mediated cell death could potentially be an avenue for dapk to regulate nematode excitotoxicity.
autophagy has a minor role in nematode excitotoxicity
the interaction between mammalian dapk and beclin <dig> is considered a major avenue for dapk’s ability to regulate autophagy  <cit> .
autophagy has been suggested to be an important factor in some neurodegenerative conditions  <cit> , and has also been shown to be a major contributor to degenerin-mediated neurodegeneration in the nematode  <cit>  .
moreover, dapk- <dig> regulates autophagy in c. elegans  <cit> .
we therefore set out to determine the role of autophagy in nematode excitotoxicity, using some of the same reagents used to demonstrate autophagy’s role in degenerin-induced neurodegeneration in the worm  <cit> .
one such reagent is the red fluorescent labeled lc <dig> –homolog lgg- <dig> .
this reporter indicates elevated autophagy by both the appearance of intracellular puncta and by an overall increase in cellular fluorescence.
we crossed imn <dig> with our excitotoxicity strain glt-3;nuis <dig>  since the red fluorescence can appear in any body cell that triggers autophagy, we used the pglr-1::gfp marker expressed in our excitotoxicity strains to focus our attention on the postsynaptic neurons that are at risk for neurodegeneration.
if autophagy is a strong component of excitotoxicity, we would expect at-risk neurons  to express autophagy marker  upon exposure to high concentrations of glu .
we counted the number of at-risk neurons that show lgg- <dig> puncta and measured the overall intensity of dsred::lgg- <dig> signal in these neurons.
since autophagy was reported to have a role in the low-level neurodegeneration caused by nuis <dig> alone  <cit> , we concentrated on the added effect of excitotoxicity by comparing dsred::lgg- <dig> signals in animals that express only the sensitizing construct nuis <dig> to those of animals in which neurons are subjected to the full excitotoxic insult .
we found that excitotoxicity resulted in only a small and statistically-insignificant increase in lgg- <dig> puncta, and no change in overall lgg- <dig> intensity in at-risk neurons .figure  <dig> a dsred::lgg- <dig> reporter of autophagy does not provide convincing evidence for triggering of autophagy in at-risk neurons exposed to the excitotoxic insult.
a) representative images showing at-risk neurons in green  and dsred::lgg- <dig> expressing cells in red.
lateral view, anterior left, dorsal up, illustration on the left describing the results expected from a putative involvement of autophagy in excitotoxicity.
expression of green labeling in the pharynx comes from the co-injection marker for the dsred::lgg- <dig> label, expressing p
myo-2
::gfp.
b) analysis of images taken from the two groups shows a similar number of at-risk neurons  showing dsred::lgg- <dig> puncta.
the observed small difference is not statistically significant.
c) the average intensity of the dsred::lgg- <dig> signal in at-risk neurons  in very similar in the two groups.
to further study the possible involvement of autophagy in nematode excitotoxicity we examined the effect of inhibiting autophagy by genetic and chemical means.
while a number of mutations and drugs have been used in the past, not all of them are available to us here.
for example, bec- <dig> ko was previously used to monitor the requirement of this autophagy regulator for mec- <dig> –induced necrosis in early development  <cit> .
however, the lethal effect of this mutation in later development, when most of the nematode excitotoxic necrosis occurs, prevents us from using this approach.
similarly, the vacuolar-type atpase inhibitor bafilomycin is commonly used to block autophagy by elevating lysosomal ph  <cit> .
however, the same v-atpase is used in neurons to acidify synaptic vesicles as a means to provide the driving force for neurotransmitter loading  <cit> , and therefore using bafilomycin can be expected to reduce neurotransmitter release.
we therefore turned to use other means of intervention that are more compatible with our system: a mutation in the autophagy regulator unc- <dig>  and the chemical inhibitor 3ma .
we noticed only a moderate effect for these two factors, evident in some developmental stages .
such a moderate effect is in line with the reported effect of unc- <dig> on nuis <dig> alone  <cit>  .
we then used an independent set of experiments and epistasis analysis to determine if this moderate effect works independently of dapk- <dig> or in the same pathway.
we noticed that the effect of blocking autophagy on the extent of excitotoxicity is reproducible only in one developmental stage .
trying to determine if dapk- <dig> and 3ma work in the same pathway, we compared their observed combined effect  to the calculated expected effect if these two processes were completely independent.
however, given the moderate size of the 3ma effect and the inherent variability in our experiments, it is currently difficult to determine if dapk- <dig> and autophagy work in the same pathway or independently.
nonetheless, the fact that the effect of autophagy is much more limited in size and duration than that of dapk- <dig> supported a continued search for other mechanisms by which dapk- <dig> might regulate excitotoxicity.figure  <dig> treatments that block autophagy  have a reproducible but small effect in nematode excitotoxicity.
a) a mutation in unc- <dig> shows a moderate effect on nematode excitotoxicity.
*p <  <dig>  ; ***p <  <dig>  b) treatment with the autophagy-blocking drug 3ma has a moderate effect on nematode excitotoxicity.
***p <  <dig>  c) independent repetition of the experiment shown in b with the addition of epistasis analysis.
the data shows that the only reproducible effect of autophagy blockade is in l <dig>  although at this stage the combined effect of autophagy blockade and dapk- <dig> correlates with a model of independent action of these two factors, the moderate extent of effects limits the strength of such a conclusion.
the dapk interaction-partner and phosphorylation-dependent peptidyl-prolyl isomerase pin1/pinn- <dig> is a conserved suppressor of neurodegeneration
pin <dig> is an isomerase that changes the conformation of proline residues located next to phosphorylated ser or thr residues, thus changing overall protein conformation and controlling the activity of many phosphoproteins  <cit> .
in recent years this protein has gained recognition as a major regulator of many signaling cascades, involved in both normal cell physiology, pathology, and in neurodegenerative diseases  <cit> .
pin <dig> is expressed in dendrites, its activity is modulated by glu signaling, and it regulates pkcζ and pkmζ  <cit> .
pin <dig> is also known for its regulation of neuronal cytoskeleton and tau protein phosphorylation, and for modulating neurodegeneration  <cit> .
recently, pin <dig> was shown to functionally interact with dapk  <cit> .
we find that pinn- <dig> ko  <cit>  causes increased neurodegeneration in nematode excitotoxicity .
like dapk- <dig> ko, the effect of pinn- <dig> ko is seen in all developmental stages , and does not seem to involve a change in glu synaptic strength , suggesting that they influence cell-death processes subsequent to-  glur.figure  <dig>
pinn- <dig> is an important suppressor of nematode excitotoxicity that does not affect basic synaptic strength.
a)
pinn- <dig> mutation enhances excitotoxicity throughout development.
**p <  <dig>  b & c)
pinn- <dig> does not affect the duration of spontaneous forward mobility or nose touch sensitivity, two sensitive measures of glu synaptic strength.
we wished to confirm that the effects of dapk- <dig> and pinn- <dig> on the dynamics of vacuolar appearance  translate to ultimate survival of specific neurons in adult animals.
given the stochastic nature of neurodegeneration in glt-3;nuis <dig> animals, it is usually difficult to identify which of the ~ <dig> at-risk head neurons are degenerating in different animals.
to circumvent this difficulty, we address the survival of specific neurons by focusing on the easily identifiable rig neurons.
the rig neurons are part of the group of at-risk head neurons, they show only very minor levels of neurodegeneration in nuis <dig> alone, and they are sensitive to all the treatments that modify total head neuron degeneration analyzed in our previous studies  <cit> .
indeed, rig neurons exhibit inverse correlation between the number of vacuole-like structures they show during development  and the number of gfp-labeled rig neurons surviving in the adult  <cit> .
we now confirm that the number of degenerating rig neurons during development is reduced by dapk- <dig> ko and increased by pinn- <dig> ko .
importantly, we find that the number of gfp-labeled rig neurons that survive to adulthood is increased by dapk- <dig> ko and decreased by pinn- <dig> ko .
these observations suggest that the effects of dapk- <dig> and pinn- <dig> on vacuolar appearance during development indeed translate to changes in ultimate survival of identified neurons.figure  <dig>
dapk- <dig> might cooperate with pinn- <dig> to regulate nematode excitotoxicity.
a) epistasis analysis testing the involvement of dapk- <dig> and pinn- <dig> in nematode excitotoxicity.
since the combined mutant shows strong suppression of neurodegeneration compared to the starting strain, these observations strongly suggest that pinn- <dig> is not downstream of dapk- <dig> .
b) verification that the effects of dapk- <dig> and pinn- <dig> mutations on the dynamics of overall neurodegeneration in head neurons translates to survival of identified neurons in the adult .
c) one of the likely models that can account for our observations on the role of dapk- <dig>  pinn- <dig>  and autophagy in nematode excitotoxicity.
finally, we wanted to determine if dapk- <dig>  and pinn- <dig>  work in the same or separate pathways.
mammalian studies suggest that dapk is acting upstream of pin <dig> to inhibit its function  <cit> , so that ko of pinn- <dig> could be expected to exert its death-stimulating effect regardless of whether dapk- <dig> is present or not.
instead, using epistasis analysis , we observe that the double knockout dapk- <dig> ; pinn- <dig> exhibited suppression of excitotoxicity.
we tried to further determine if the effect of dapk-1; pinn- <dig> double knockout reflects an intermediate outcome  or a dapk- <dig> ko –only-like outcome .
to that end we calculated what would be the effect of these two factors acting in parallel, to predict their cumulative independent effect .
unfortunately, the small difference between the observed effect of dapk- <dig> alone and the calculated independent cumulative effects of dapk- <dig> and pinn- <dig>  together with the variability in our data, do not allow us to discriminate with confidence between these two options.
we therefore limit our conclusion to say that dapk- <dig> acts either downstream or in parallel to-  pinn- <dig> in nematode excitotoxicity.
discussion
mammalian studies have generated a plethora of proposed mechanisms in excitotoxicity, and some of these studies include suggested pathways for the involvement of dapk in critical cell death events.
however, the significance of these proposed mechanisms across divergent excitotoxic conditions remains unclear.
in our study we focused on a glutamate-dependent neuronal death in c. elegans and examined a set of candidate mechanisms to define those that might be conserved through a large evolutionary distance.
we previously found that some core constitutes of excitotoxicity are well conserved in our nematode model of glut ko –triggered and cpar-mediated neuronal necrosis.
these include both death-promoting factors , and neuroprotective factors .
we now report that dapk is also a highly conserved regulator of excitotoxicity , though we note that dapk- <dig> ko does not bring neurodegeneration all the way down to background levels.
we find no evidence for dapk- <dig> –mediated regulation of synaptic strength, as defined by the spontaneous mobility assay , suggesting that the effect of dapk on mammalian nr2b/glun2b, though probably very important, does not extend to all forms of excitotoxicity.
we also found no role for the danger-related protein mab- <dig> in nematode excitotoxicity .
although dapk- <dig> is an important regulator of autophagy in c. elegans, we find only a minor role for autophagy in the neurodegenerative condition we study in glt-3;nuis <dig> animals.
we find that blocking autophagy has a relatively small neurodegeneration-reducing effect .
this effect is smaller than the neurodegeneration-suppressing effect of dapk- <dig> ko .
therefore, even if related, autophagy cannot account for the majority of dapk’s effect in glt-3;nuis <dig> animals.
the minor effect of autophagy on neurodegeneration in glt-3;nuis <dig> is in sharp contrast to the major effect that autophagy has on degenerin-mediated neurodegeneration, where it suppresses neurodegeneration by 75-90%  <cit> .
we further note that these previous studies have demonstrated that blocking autophagy decreases the extent of low-level neurodegeneration triggered by nuis <dig> alone  <cit> .
since neurodegeneration by nuis <dig> alone  is part of the total number of degenerating neurons we count in our assay , the small effect of autophagy seen in the current study can be attributed to its documented effect of nuis <dig> alone.
indeed, we see no significant difference in labeling of fluorescent autophagy reporter when comparing nuis <dig> to glt-3;nuis <dig> animals .
the results of epistasis analysis are not conclusive, but put together with the difference in size and timing of the effects of dapk- <dig> and autophagy on nematode excitotoxicity, our observations cause us to favor one of the possible models in which autophagy has only a minor role in nematode excitotoxicity in parallel to the role of dapk- <dig> .
we emphasize that the parallel action of dapk and autophagy is preferred by us in the specific scenario of nematode excitotoxic stress, while other stresses might include dapk and autophagy in other pathway configurations.
the observation that the dapk-interaction-partner pin1/pinn- <dig> is a significant regulator of excitotoxicity is particularly intriguing and novel.
in addition to its involvement in cancer  <cit>  and stress response  <cit> , pin <dig> has been shown to be a critical regulator of dendritic glu responses  <cit>  and of neurodegeneration in alzheimer disease  <cit> .
the results of our epistasis analysis do not seem to support a role for pin1/pinn- <dig> as an obligatory step downstream of dapk/dapk- <dig>  as might be inferred from mammalian cancer studies  <cit> .
instead, our data supports that pinn- <dig> acts in parallel or upstream of dapk- <dig> .
CONCLUSIONS
put together, our study suggests that nematode excitotoxicity can be an important tool to sift through many proposed mechanisms of excitotoxicity, allowing us to identify conserved mechanisms that might be at the core of neurodegenerative processes common across divergent excitotoxic scenarios.
furthermore, our studies now illuminate dapk/dapk- <dig> and pin1/pinn- <dig> as two such factors, important for excitotoxicity mechanisms across a large evolutionary distance.
we can now further use this system to decipher dapk- <dig> and pinn- <dig> –related mechanisms in nematode excitotoxicity, with the hope that such understanding might help us to continue uncovering conserved core mechanisms in this important form of neurodegeneration.

 excitotoxicity is a neurodegenerative process believed to be the central mediator of brain damage in acute conditions such as brain ischemia and traumatic injury, and an important contributor to a range of chronic neurodegenerative diseases. in excitotoxicity, the malfunction of glutamate transporters causes accumulation of glu in excitatory synapses and exaggerated stimulation of postsynaptic glu receptors. the excessive influx of ions into the postsynaptic neurons leads to their cell death via a spectrum of mechanisms that range from necrosis to apoptosis or even recovery. despite our familiarity with the first few steps in excitotoxicity, our understanding of the steps following ca2+ influx is very limited. clinical trials using glurs antagonists ended with disappointment, and recent data suggests that using antagonists to block glur functions might be counterproductive, because glu signaling includes both neurotoxic and pro-survival cascades. these complications emphasize the need to illuminate cell-death-specific signaling cascades in excitotoxicity downstream of glurs. a considerable number of such excitotoxic mechanisms have been suggested, but in many cases the data that supports a given suggestion is limited to specific excitotoxic paradigms. one suggested mediator of excitotoxicity implicated in multiple experimental setups is the cam-dependent death associated protein kinase. originally identified by an unbiased screen for mediators of interferon-induced cell death, dapk was later recognized as a molecular switch that controls the choice between cell death processes such as apoptosis and autophagy. moreover, an elaborate web of biochemical interactions and functional connections has been revealed, placing dapk in a key position at the center of many critical signaling cascades. a considerable body of evidence suggests that dapk also contributes to cell death in excitotoxicity, and its inhibition reduces neuronal loss in models of brain ischemia. several mechanisms have been suggested to explain dapk’s involvement in excitotoxicity, but the issue remains controversial. one possible mechanism involves dapk’s regulation of autophagy, a process that modulates some neurodegenerative conditions. alternatively, an intriguing study suggests the involvement of danger, a protein that contains a region of homology to the nematode protein mab- and functions as a regulator of the ip3r. indeed danger was found to also interact with and inhibit the activity of dapk. the most cited suggestion in the field attributes the involvement of dapk in excitotoxicity to the potentiation of ca2+ currents through nr2b/glun2b subunit-containing complexes of the nmda- receptor family of glurs. this suggestion fits well with a proposed leading role for extrasynaptic nr2b/glun2b –containing nmda-rs in excitotoxicity. however, recently the proposed unique significance of nr2b/glun2b –containing extrasynaptic nmda-rs in excitotoxicity has been brought into question. moreover, an earlier study suggests that in some mammalian neurons dapk knockout provides protection from excitotoxicity that is not dependent on nmda-rs. indeed, some cases of excitotoxicity are mediated by another family of glurs, the ca2+ -permeable ampa receptors. these observations suggest that if dapk is widely involved in excitotoxicity, including in cases where nr2b/glun2b is not a main determinant of neurodegeneration, it might act through additional mechanisms to exert its effect. we hypothesize that many signaling cascades might be involved in specific cases of excitotoxicity, depending on the exact scenario being used to induce it. however, the key features that constitute the core of the excitotoxic process might be conserved across differences in cell death scenarios and large evolutionary distances, as is the case in apoptosis and autophagy. we therefore set out to study the role of dapk in excitotoxicity in our model of cpar-mediated neurodegeneration in c. elegans, where glur-dependent necrosis of central neurons postsynaptic to glu connections is triggered by knockout of the glut gene glt- in a sensitized background. indeed, this model has proven effective in identifying core processes that are conserved between nematode and mammalian excitotoxicity. dapk is particularly well conserved in c. elegans. the nematode dapk- is widely expressed, allowing us to test its involvement in nematode excitotoxicity and to study its mechanism of action. in this study we establish the central role of dapk in glu-triggered neurodegeneration in c. elegans, suggesting that its function is conserved across evolution and excitotoxic scenarios. we find little or no support for the views that dapk’s regulation of excitotoxicity is mediated through the modulation of synaptic strength, mab- or autophagy. instead, we identify pinn- the nematode homolog of the dapk-interaction-partner and phosphorylation-dependent peptidyl-prolyl isomerase pin as an important factor in nematode excitotoxicity.  dapk- has a central role in nematode excitotoxicity to test the involvement of dapk in nematode excitotoxicity we combined our excitotoxicity strain with a deletion allele of the nematode homolog of dapk, dapk- a gene knockout that was used to confirm the effect of the nematode dapk in autophagy and innate immunity. in our previously described model of neurodegeneration we use the sensitizing transgenic modification nuis where hyperactive gαs and gfp are expressed under the glr- promoter in ~ neurons and cause glur-independent stochastic degeneration of ~ of these at-risk neurons per animal. when we add the ko of the glut gene glt- we observe that more of these at-risk neurons degenerate. the glut-ko-triggered exacerbated necrosis in glt-3;nuis is glur-dependent, and therefore qualifies as nematode excitotoxicity. nematode excitotoxicity causes neuronal swelling and death that is manifested with characteristic kinetics as gradually and stochastically appearing vacuole-like structures in some of the at-risk postsynaptic neurons. these vacuole-like structures become more abundant during larval development as the glu signaling system matures, and then decline due to removal of cell corpses by engulfment. we now observe that adding dapk- ko to this excitotoxicity strain causes a strong and statistically significant suppression of neurodegeneration throughout development. to further confirm the contribution of dapk- to nematode excitotoxicity, we overexpressed the wt dapk- cdna from an extra-chromosomal transgenic construct under a heat-shock promoter. since heat-shock might affect susceptibility to neurodegeneration, we took special care to compare an exact match of treated animals, without or with the dapk- overexpression transgene. to that end we took advantage of the fact that the random and partial segregation of the non-integrated overexpression construct allows us to compare transgenic and non-transgenic animals on the same plate exposed to the same conditions. we observed that dapk- overexpression resulted in a strong and statistically significant potentiation of necrotic neurodegeneration in postsynaptic neurons in all developmental stages. together, our data indicate that dapk is an important mediator of excitotoxicity in c. elegans, suggesting that diverse scenarios of excitotoxicity share a common mechanism that assigns a central role to dapk.figure dapk- is an important mediator of nematode excitotoxicity. a) dynamics of neurodegeneration in nematode excitotoxicity during development. dapk- ko mutation suppresses neurodegeneration in all developmental stages, but does not bring it down to background levels. b) overexpression of wt dapk- from a heat-shock promoter enhances neurodegeneration compared to matched controls. in all bar graphs, error bars represent se. statistical significance is calculated using z score. *p < ; ***p < . dapk- ko does not alter presynaptic release or postsynaptic glu response we used two main guidelines in trying to track the mechanism by which dapk- regulates excitotoxic neurodegeneration in the nematode: 1) we looked at previous reports suggesting specific mechanisms for dapk’s involvement in mammalian excitotoxicity; 2) we inferred from the general map of dapk’s connectome in other cell processes which additional proteins are plausible candidates for mediating dapk’s effect in nematode excitotoxicity. one line of evidence suggests that dapk interacts with, and may regulate the function of, syntaxin 1a. syntaxin is part of the general mechanism of vesicular neurotransmitter release, a mechanism that is shared among all neurotransmitters. therefore, an effect of dapk- ko on any component of the synaptic vesicle release could lead to dapk-mediated changes in excitotoxicity levels. however, given the ubiquitous expression of this gene, such an effect of dapk on the common synaptic vesicle release mechanism will affect the dynamics of neurotransmitter release in all synapses. the study of synaptic vesicle release is very well developed in the nematode, and aldicarb is routinely used in c. elegans to identify mutations that cause even modest changes to the general synaptic release mechanism. as aldicarb suppresses the degradation of acetylcholine in the neuro-muscular junction, it causes animal paralysis with a typical dynamics. mutations that reduce the activity of the general, common vesicle release mechanism cause a pronounced resistance to aldicarb, while mutations that enhance synaptic release cause increased sensitivity to aldicarb. we observed that the sensitivity of dapk- animals to aldicarb is indistinguishable from that of wt animals, suggesting that dapk- ko does not modify the synaptic release mechanism, as would have been expected of an effect on syntaxin.figure the effect of dapk- does not correlate with several proposed mechanisms. a) aldicarb assay measures the overall activity of the common mechanism of synaptic vesicle release. complexin mutant is used as example of an oversensitive mutant, while rab- serves as an example of aldicarb resistant mutant. dapk- mutants do not show oversensitivity or resistance, suggesting their level of synaptic release is roughly normal. b) duration of forward runs during spontaneous mobility is a sensitive reporter of the overall strength of glu synapses that control this parameter. even small changes in the activity or number of glurs typically modify this behavior. dapk- mutants are not different from normal counterparts in either a wt background, in an excessive glu stimulation background, or in the sensitized background used in our excitotoxicity model. these three conditions by themselves do not cause excessive command neuron loss, a loss that might otherwise render the assay uninformative as it is suppressible by dapk- c) a mutation in the gene encoding the danger-related protein mab- does not affect nematode excitotoxicity. d) a mutation in the gene encoding the nematode homolog of camkk does not affect nematode excitotoxicity. we next asked if dapk modifies the extent of postsynaptic response in the glutamatergic synapses where excitotoxicity occurs. two behavioral assays have proven very effective in detecting even small changes in the activity level of glutamatergic synapses in c. elegans. these are the nose touch assay, and the duration of spontaneous forward mobility assay. therefore, if dapk- ko causes changes in the specific glu packaging mechanism in synaptic vesicles, or changes the number or activity-level of glurs in the synapse, this should be reflected in behavioral changes in these assays. in the particular case of dapk- a secondary phenotype of this mutation, namely overgrowth of cuticle on the animal’s nose, makes the nose-touch assay less informative. however, the duration of spontaneous forward mobility depends on the internal balance between forward and backward circuits, and should not be affected by the cuticle aberration. this assay is very reliable in detecting both under-activity and over-activity of the relevant glu synapses, allowing us to use it as a proxy measure for glu synaptic strength. we did not observe any changes in spontaneous mobility triggered by dapk- ko, either in wt background or in the background of each of the two components used in our excitotoxicity model to trigger the intensified glu signaling. these observations suggest that dapk- has no strong effect on synaptic release or the overall strength of signaling in glu synapses in c. elegans. the danger-related protein mab- and the camkk ckk- do not exert strong regulation of nematode excitotoxicity the characterization of dapk as a cam-dependent kinase is particularly intriguing to us, since ca2+ signaling is critical to excitotoxicity in both mammals and nematodes. we therefore examined dapk-partners that might also be involved in ca2+ signaling. danger is a mammalian dapk inhibitor that regulates ca2+ release from the er. there is no direct, full-length homolog of danger in the worm genome, but the core of the mammalian protein is thought to be homologous to the nematode protein mab-. it is therefore reasonable to assume that mab- will be essential to the role of a hypothetic danger-like complex in c. elegans. however, a mutation in mab- had no effect on nematode excitotoxicity. another important protein that binds mammalian dapk and is involved in ca2+ signaling is camkk. however, again, a mutation in the only camkk gene in the worm, ckk-, had no effect on excitotoxicity. these results suggest that these two putative ca2+ signaling regulators and dapk-interaction-partners do not have a large contribution to nematode excitotoxicity. we also considered the many other proteins that are known to interact with dapk as possible modulators of excitotoxicity, but we could not assign to many of them high priority because either there was no immediate obvious connection to neurodegeneration, there is no clear nematode homolog, the nematode homolog is not known to be active in c. elegans neurons, or the process in which this protein is involved has been shown by us to be not involved in nematode excitotoxicity. however, dapk is also known to interact with beclin a key regulator of the evolutionary conserved process of autophagy, suggesting that autophagy-mediated cell death could potentially be an avenue for dapk to regulate nematode excitotoxicity. autophagy has a minor role in nematode excitotoxicity the interaction between mammalian dapk and beclin is considered a major avenue for dapk’s ability to regulate autophagy. autophagy has been suggested to be an important factor in some neurodegenerative conditions, and has also been shown to be a major contributor to degenerin-mediated neurodegeneration in the nematode. moreover, dapk- regulates autophagy in c. elegans. we therefore set out to determine the role of autophagy in nematode excitotoxicity, using some of the same reagents used to demonstrate autophagy’s role in degenerin-induced neurodegeneration in the worm. one such reagent is the red fluorescent labeled lc –homolog lgg-. this reporter indicates elevated autophagy by both the appearance of intracellular puncta and by an overall increase in cellular fluorescence. we crossed imn with our excitotoxicity strain glt-3;nuis since the red fluorescence can appear in any body cell that triggers autophagy, we used the pglr-1::gfp marker expressed in our excitotoxicity strains to focus our attention on the postsynaptic neurons that are at risk for neurodegeneration. if autophagy is a strong component of excitotoxicity, we would expect at-risk neurons to express autophagy marker upon exposure to high concentrations of glu. we counted the number of at-risk neurons that show lgg- puncta and measured the overall intensity of dsred::lgg- signal in these neurons. since autophagy was reported to have a role in the low-level neurodegeneration caused by nuis alone, we concentrated on the added effect of excitotoxicity by comparing dsred::lgg- signals in animals that express only the sensitizing construct nuis to those of animals in which neurons are subjected to the full excitotoxic insult. we found that excitotoxicity resulted in only a small and statistically-insignificant increase in lgg- puncta, and no change in overall lgg- intensity in at-risk neurons.figure a dsred::lgg- reporter of autophagy does not provide convincing evidence for triggering of autophagy in at-risk neurons exposed to the excitotoxic insult. a) representative images showing at-risk neurons in green and dsred::lgg- expressing cells in red. lateral view, anterior left, dorsal up, illustration on the left describing the results expected from a putative involvement of autophagy in excitotoxicity. expression of green labeling in the pharynx comes from the co-injection marker for the dsred::lgg- label, expressing p myo-2::gfp. b) analysis of images taken from the two groups shows a similar number of at-risk neurons showing dsred::lgg- puncta. the observed small difference is not statistically significant. c) the average intensity of the dsred::lgg- signal in at-risk neurons in very similar in the two groups. to further study the possible involvement of autophagy in nematode excitotoxicity we examined the effect of inhibiting autophagy by genetic and chemical means. while a number of mutations and drugs have been used in the past, not all of them are available to us here. for example, bec- ko was previously used to monitor the requirement of this autophagy regulator for mec- –induced necrosis in early development. however, the lethal effect of this mutation in later development, when most of the nematode excitotoxic necrosis occurs, prevents us from using this approach. similarly, the vacuolar-type atpase inhibitor bafilomycin is commonly used to block autophagy by elevating lysosomal ph. however, the same v-atpase is used in neurons to acidify synaptic vesicles as a means to provide the driving force for neurotransmitter loading, and therefore using bafilomycin can be expected to reduce neurotransmitter release. we therefore turned to use other means of intervention that are more compatible with our system: a mutation in the autophagy regulator unc- and the chemical inhibitor 3ma. we noticed only a moderate effect for these two factors, evident in some developmental stages. such a moderate effect is in line with the reported effect of unc- on nuis alone. we then used an independent set of experiments and epistasis analysis to determine if this moderate effect works independently of dapk- or in the same pathway. we noticed that the effect of blocking autophagy on the extent of excitotoxicity is reproducible only in one developmental stage. trying to determine if dapk- and 3ma work in the same pathway, we compared their observed combined effect to the calculated expected effect if these two processes were completely independent. however, given the moderate size of the 3ma effect and the inherent variability in our experiments, it is currently difficult to determine if dapk- and autophagy work in the same pathway or independently. nonetheless, the fact that the effect of autophagy is much more limited in size and duration than that of dapk- supported a continued search for other mechanisms by which dapk- might regulate excitotoxicity.figure treatments that block autophagy have a reproducible but small effect in nematode excitotoxicity. a) a mutation in unc- shows a moderate effect on nematode excitotoxicity. *p < ; ***p <  b) treatment with the autophagy-blocking drug 3ma has a moderate effect on nematode excitotoxicity. ***p <  c) independent repetition of the experiment shown in b with the addition of epistasis analysis. the data shows that the only reproducible effect of autophagy blockade is in l although at this stage the combined effect of autophagy blockade and dapk- correlates with a model of independent action of these two factors, the moderate extent of effects limits the strength of such a conclusion. the dapk interaction-partner and phosphorylation-dependent peptidyl-prolyl isomerase pin1/pinn- is a conserved suppressor of neurodegeneration pin is an isomerase that changes the conformation of proline residues located next to phosphorylated ser or thr residues, thus changing overall protein conformation and controlling the activity of many phosphoproteins. in recent years this protein has gained recognition as a major regulator of many signaling cascades, involved in both normal cell physiology, pathology, and in neurodegenerative diseases. pin is expressed in dendrites, its activity is modulated by glu signaling, and it regulates pkcζ and pkmζ. pin is also known for its regulation of neuronal cytoskeleton and tau protein phosphorylation, and for modulating neurodegeneration. recently, pin was shown to functionally interact with dapk. we find that pinn- ko causes increased neurodegeneration in nematode excitotoxicity. like dapk- ko, the effect of pinn- ko is seen in all developmental stages, and does not seem to involve a change in glu synaptic strength, suggesting that they influence cell-death processes subsequent to- glur.figure pinn- is an important suppressor of nematode excitotoxicity that does not affect basic synaptic strength. a) pinn- mutation enhances excitotoxicity throughout development. **p <  b & c) pinn- does not affect the duration of spontaneous forward mobility or nose touch sensitivity, two sensitive measures of glu synaptic strength. we wished to confirm that the effects of dapk- and pinn- on the dynamics of vacuolar appearance translate to ultimate survival of specific neurons in adult animals. given the stochastic nature of neurodegeneration in glt-3;nuis animals, it is usually difficult to identify which of the ~ at-risk head neurons are degenerating in different animals. to circumvent this difficulty, we address the survival of specific neurons by focusing on the easily identifiable rig neurons. the rig neurons are part of the group of at-risk head neurons, they show only very minor levels of neurodegeneration in nuis alone, and they are sensitive to all the treatments that modify total head neuron degeneration analyzed in our previous studies. indeed, rig neurons exhibit inverse correlation between the number of vacuole-like structures they show during development and the number of gfp-labeled rig neurons surviving in the adult. we now confirm that the number of degenerating rig neurons during development is reduced by dapk- ko and increased by pinn- ko. importantly, we find that the number of gfp-labeled rig neurons that survive to adulthood is increased by dapk- ko and decreased by pinn- ko. these observations suggest that the effects of dapk- and pinn- on vacuolar appearance during development indeed translate to changes in ultimate survival of identified neurons.figure dapk- might cooperate with pinn- to regulate nematode excitotoxicity. a) epistasis analysis testing the involvement of dapk- and pinn- in nematode excitotoxicity. since the combined mutant shows strong suppression of neurodegeneration compared to the starting strain, these observations strongly suggest that pinn- is not downstream of dapk-. b) verification that the effects of dapk- and pinn- mutations on the dynamics of overall neurodegeneration in head neurons translates to survival of identified neurons in the adult. c) one of the likely models that can account for our observations on the role of dapk- pinn- and autophagy in nematode excitotoxicity. finally, we wanted to determine if dapk- and pinn- work in the same or separate pathways. mammalian studies suggest that dapk is acting upstream of pin to inhibit its function, so that ko of pinn- could be expected to exert its death-stimulating effect regardless of whether dapk- is present or not. instead, using epistasis analysis, we observe that the double knockout dapk-; pinn- exhibited suppression of excitotoxicity. we tried to further determine if the effect of dapk-1; pinn- double knockout reflects an intermediate outcome or a dapk- ko –only-like outcome. to that end we calculated what would be the effect of these two factors acting in parallel, to predict their cumulative independent effect. unfortunately, the small difference between the observed effect of dapk- alone and the calculated independent cumulative effects of dapk- and pinn- together with the variability in our data, do not allow us to discriminate with confidence between these two options. we therefore limit our conclusion to say that dapk- acts either downstream or in parallel to- pinn- in nematode excitotoxicity. discussion mammalian studies have generated a plethora of proposed mechanisms in excitotoxicity, and some of these studies include suggested pathways for the involvement of dapk in critical cell death events. however, the significance of these proposed mechanisms across divergent excitotoxic conditions remains unclear. in our study we focused on a glutamate-dependent neuronal death in c. elegans and examined a set of candidate mechanisms to define those that might be conserved through a large evolutionary distance. we previously found that some core constitutes of excitotoxicity are well conserved in our nematode model of glut ko –triggered and cpar-mediated neuronal necrosis. these include both death-promoting factors, and neuroprotective factors. we now report that dapk is also a highly conserved regulator of excitotoxicity, though we note that dapk- ko does not bring neurodegeneration all the way down to background levels. we find no evidence for dapk- –mediated regulation of synaptic strength, as defined by the spontaneous mobility assay, suggesting that the effect of dapk on mammalian nr2b/glun2b, though probably very important, does not extend to all forms of excitotoxicity. we also found no role for the danger-related protein mab- in nematode excitotoxicity. although dapk- is an important regulator of autophagy in c. elegans, we find only a minor role for autophagy in the neurodegenerative condition we study in glt-3;nuis animals. we find that blocking autophagy has a relatively small neurodegeneration-reducing effect. this effect is smaller than the neurodegeneration-suppressing effect of dapk- ko. therefore, even if related, autophagy cannot account for the majority of dapk’s effect in glt-3;nuis animals. the minor effect of autophagy on neurodegeneration in glt-3;nuis is in sharp contrast to the major effect that autophagy has on degenerin-mediated neurodegeneration, where it suppresses neurodegeneration by 75-90%. we further note that these previous studies have demonstrated that blocking autophagy decreases the extent of low-level neurodegeneration triggered by nuis alone. since neurodegeneration by nuis alone is part of the total number of degenerating neurons we count in our assay, the small effect of autophagy seen in the current study can be attributed to its documented effect of nuis alone. indeed, we see no significant difference in labeling of fluorescent autophagy reporter when comparing nuis to glt-3;nuis animals. the results of epistasis analysis are not conclusive, but put together with the difference in size and timing of the effects of dapk- and autophagy on nematode excitotoxicity, our observations cause us to favor one of the possible models in which autophagy has only a minor role in nematode excitotoxicity in parallel to the role of dapk-. we emphasize that the parallel action of dapk and autophagy is preferred by us in the specific scenario of nematode excitotoxic stress, while other stresses might include dapk and autophagy in other pathway configurations. the observation that the dapk-interaction-partner pin1/pinn- is a significant regulator of excitotoxicity is particularly intriguing and novel. in addition to its involvement in cancer and stress response, pin has been shown to be a critical regulator of dendritic glu responses and of neurodegeneration in alzheimer disease. the results of our epistasis analysis do not seem to support a role for pin1/pinn- as an obligatory step downstream of dapk/dapk- as might be inferred from mammalian cancer studies. instead, our data supports that pinn- acts in parallel or upstream of dapk-.  put together, our study suggests that nematode excitotoxicity can be an important tool to sift through many proposed mechanisms of excitotoxicity, allowing us to identify conserved mechanisms that might be at the core of neurodegenerative processes common across divergent excitotoxic scenarios. furthermore, our studies now illuminate dapk/dapk- and pin1/pinn- as two such factors, important for excitotoxicity mechanisms across a large evolutionary distance. we can now further use this system to decipher dapk- and pinn- –related mechanisms in nematode excitotoxicity, with the hope that such understanding might help us to continue uncovering conserved core mechanisms in this important form of neurodegeneration. 
our studies highlight the prominence of dapk and pin1/pinn- as conserved mediators of cell death processes in diverse scenarios of neurodegeneration. here we analyze a well-characterized model of excitotoxicity in the nematode c. elegans to show that dapk is an important mediator of excitotoxic neurodegeneration across a large evolutionary distance. in contrast, pin1/pinn- suppresses neurodegeneration in our excitotoxicity model. we further show that some proposed mechanisms of dapk’s action do not have a major role in nematode excitotoxicity. excitotoxicity is a central process in widespread neurodegenerative conditions such as brain ischemia and chronic neurological diseases.
 excitotoxicity is a central process in widespread neurodegenerative conditions such as brain ischemia and chronic neurological diseases. many mechanisms have been suggested to mediate excitotoxicity, but their significance across diverse excitotoxic scenarios remains unclear. death associated protein kinase, a critical molecular switch that controls a range of key signaling and cell death pathways, has been suggested to have an important role in excitotoxicity. however, the molecular mechanism by which dapk exerts its effect is controversial. a few distinct mechanisms have been suggested by single studies, and a larger array of potential mechanisms is implicated by the extensive interactome of dapk.  here we analyze a well-characterized model of excitotoxicity in the nematode c. elegans to show that dapk is an important mediator of excitotoxic neurodegeneration across a large evolutionary distance. we further show that some proposed mechanisms of dapk’s action do not have a major role in nematode excitotoxicity. in contrast, pin1/pinn- suppresses neurodegeneration in our excitotoxicity model.  our studies highlight the prominence of dapk and pin1/pinn- as conserved mediators of cell death processes in diverse scenarios of neurodegeneration. keywords ischemiaglutamateexcitotoxicityneurodegenerationdeath-associated protein kinaseautophagypeptidyl prolyl isomerase pin1issue-copyright-statement© the author 2015 