- Research article
- Open Access
Death Associated Protein Kinase (DAPK) -mediated neurodegenerative mechanisms in nematode excitotoxicity
© Del Rosario et al.; licensee BioMed Central. 2015
Received: 1 October 2014
Accepted: 31 March 2015
Published: 23 April 2015
Excitotoxicity (the toxic overstimulation of neurons by the excitatory transmitter Glutamate) 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 (DAPK), 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 (sometimes contradicting) 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 (modulation of synaptic strength, involvement of the DANGER-related protein MAB-21, and autophagy) do not have a major role in nematode excitotoxicity. In contrast, Pin1/PINN-1 (a DAPK interaction-partner and a peptidyl-prolyl isomerase involved in chronic neurodegenerative conditions) suppresses neurodegeneration in our excitotoxicity model.
Our studies highlight the prominence of DAPK and Pin1/PINN-1 as conserved mediators of cell death processes in diverse scenarios 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 [1-4]. In excitotoxicity, the malfunction of Glutamate (Glu) Transporters (GluTs) [5-7] causes accumulation of Glu in excitatory synapses and exaggerated stimulation of postsynaptic Glu receptors (GluRs) . The excessive influx of ions (especially Ca2+) into the postsynaptic neurons leads to their cell death via a spectrum of mechanisms that range from necrosis (at the core of the ischemic damage) to apoptosis or even recovery (at the penumbra). 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 [9,10]. 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 (DAPK) [11-14]. 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 [16,17]. 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 [11,18]. 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-21 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 (NMDA-Rs) 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 [26-28]. 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 (CPARs) [30-32]. 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 [33,34] and autophagy [35,36]. 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 (ko) of the GluT gene glt-3  in a sensitized background (nuIs5 ). Indeed, this model has proven effective in identifying core processes that are conserved between nematode and mammalian excitotoxicity [37,40,41]. DAPK is particularly well conserved in C. elegans (in 52% sequence homology, presenting all of DAPK’s functional domains, and in its involvement in a number of signaling cascades [42-44]). The nematode DAPK-1 is widely expressed (including in neurons ), 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-21, or autophagy. Instead, we identify PINN-1, the nematode homolog of the DAPK-interaction-partner and phosphorylation-dependent peptidyl-prolyl isomerase Pin1, as an important factor in nematode excitotoxicity.
DAPK-1 has a central role in nematode excitotoxicity
dapk-1 ko does not alter presynaptic release or postsynaptic Glu response
We next asked if DAPK modifies the extent of postsynaptic response in the glutamatergic synapses where excitotoxicity occurs (as reported in mammals, where DAPK was suggested to modify synaptic strength ). 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 (NOT) assay, and the duration of spontaneous forward mobility assay. Therefore, if dapk-1 ko causes changes in the specific Glu packaging mechanism in synaptic vesicles (e.g., by affecting vGluTs), 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-1, 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 [50-52], 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-1 ko, either in WT background or in the background of each of the two components (glt-3 or nuIs5) used in our excitotoxicity model to trigger the intensified Glu signaling (Figure 2B). These observations suggest that dapk-1 has no strong effect on synaptic release or the overall strength of signaling in Glu synapses in C. elegans.
The DANGER-related protein MAB-21 and the CaMKK CKK-1 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 [22,23]. 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-21 . It is therefore reasonable to assume that mab-21 will be essential to the role of a hypothetic DANGER-like complex in C. elegans. However, a mutation in mab-21 had no effect on nematode excitotoxicity (Figure 2C). 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-1 , had no effect on excitotoxicity (Figure 2D). 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 (e.g., tropomyosin), there is no clear nematode homolog (e.g., NFκB), the nematode homolog is not known to be active in C. elegans neurons (e.g., p53), or the process in which this protein is involved has been shown by us to be not involved in nematode excitotoxicity (e.g., apoptosis ). However, DAPK is also known to interact with Beclin1, 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 DAPK interaction-partner and phosphorylation-dependent peptidyl-prolyl isomerase Pin1/PINN-1 is a conserved suppressor of neurodegeneration
Finally, we wanted to determine if dapk-1 (where a KO causes decreased neurodegeneration) and pinn-1 (where a KO causes increased neurodegeneration) work in the same or separate pathways. Mammalian studies suggest that DAPK is acting upstream of Pin1 to inhibit its function , so that KO of pinn-1 could be expected to exert its death-stimulating effect regardless of whether dapk-1 is present or not. Instead, using epistasis analysis (Figure 6A), we observe that the double knockout dapk-1 ; pinn-1 exhibited suppression of excitotoxicity. We tried to further determine if the effect of dapk-1; pinn-1 double knockout reflects an intermediate outcome (in line with an independent, parallel effect of these two factors) or a dapk-1 ko –only-like outcome (in line with an obligatory sequential effect, where the dapk-1 mutation-induced decrease in neurodegeneration completely masks the ability of pinn-1 mutation to increase neurodegeneration). To that end we calculated what would be the effect of these two factors acting in parallel, to predict their cumulative independent effect (Figure 6A). Unfortunately, the small difference between the observed effect of dapk-1 alone and the calculated independent cumulative effects of dapk-1 and pinn-1, 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-1 acts either downstream or in parallel to- (but not upstream of-) pinn-1 in nematode excitotoxicity.
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 (such as release of Ca2+ from the ER ), and neuroprotective factors (such as cell stress resistance to insults, resistance that is conferred by FoxO/DAF-16 [40,41]). We now report that DAPK is also a highly conserved regulator of excitotoxicity (Figure 1A and B), though we note that dapk-1 ko does not bring neurodegeneration all the way down to background levels. We find no evidence for dapk-1 –mediated regulation of synaptic strength, as defined by the spontaneous mobility assay (Figure 2B), 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-21 in nematode excitotoxicity (though DANGER is a much larger protein than MAB-21, suggesting it might have additional functions not tested here).
Although dapk-1 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;nuIs5 animals. We find that blocking autophagy has a relatively small neurodegeneration-reducing effect (~0.5-1 dying neuron/animal, Figure 4). This effect is smaller than the neurodegeneration-suppressing effect of dapk-1 ko (a decrease of ~2.5 dying neurons/animal, three independent isolates counted in Figures 1A, 4C, and 6A). Therefore, even if related, autophagy cannot account for the majority of DAPK’s effect in glt-3;nuIs5 animals. The minor effect of autophagy on neurodegeneration in glt-3;nuIs5 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 nuIs5 alone . Since neurodegeneration by nuIs5 alone (independent of excitotoxicity) is part of the total number of degenerating neurons we count in our assay (accounting for ~1 dying neuron/animal ), the small effect of autophagy seen in the current study can be attributed to its documented effect of nuIs5 alone. Indeed, we see no significant difference in labeling of fluorescent autophagy reporter when comparing nuIs5 to glt-3;nuIs5 animals (Figure 3). The results of epistasis analysis are not conclusive, but put together with the difference in size and timing of the effects of dapk-1 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-1 (Figure 6C). 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-1 is a significant regulator of excitotoxicity is particularly intriguing and novel. In addition to its involvement in cancer  and stress response , Pin1 has been shown to be a critical regulator of dendritic Glu responses [72,73] and of neurodegeneration in Alzheimer disease [69,74,75]. The results of our epistasis analysis do not seem to support a role for Pin1/PINN-1 as an obligatory step downstream of DAPK/DAPK-1, as might be inferred from mammalian cancer studies . Instead, our data supports that PINN-1 acts in parallel or upstream of DAPK-1 (Figure 6A and C).
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-1 and Pin1/PINN-1 as two such factors, important for excitotoxicity mechanisms across a large evolutionary distance. We can now further use this system to decipher DAPK-1 and PINN-1 –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.
The following C. elegans strains were obtained from the C. elegans Genetic Center (CGC), from the Japanese National Bioresource Project (NBSP), or from the original producers: WT: Bristol N2 (RRID:CGC_N2 (ancestral)); Nematode excitotoxicity model: ZB1102: glt-3(bz34) IV; nuIs5 V; (RRID:CGC_ZB1102) dapk-1 : VC432: dapk-1(gk219) I; (RRID:CGC_VC432) dapk-1 over-expression: CZ9277: frIs7[P nlp-29 ::GFP; P col-12 :DsRed] (IV); juEx1933[P hsp16 ::DAPK-1; P ttx-3 ::RFP] (the frIs7 insertion was later eliminated during our cross); Aldicarb assay control strains: sv-hyper-releasing/aldicarb-oversensitive RB1367: cpx-1(ok1552) (RRID:CGC_RB1367); sv-under-releasing/aldicarb-resistant NM791: rab-3(js49) (RRID:CGC_NM791): Autophagy modulator unc-51: CB369: unc-51(e369) V (RRID:CGC_CB369); Autophagy label, DsRed::LGG-1: (originally created by the Tavernarakis lab , strain naming here as per BMC policy) IMN21: N2; Ex [P lgg-1 :: DsRed:: LGG-1; P myo-2 :: GFP]; Glu-regulated mobility defective control: VM1268: nmr-1(ak4) II; glr-2(ak10) glr-1(ky176) III; DANGER-related protein MAB-21: EM128: mab-21(bx53) III (Q203* , CAG -- > UAG) (RRID:CGC_EM128). CaMKK-like KO: ckk-1(ok1033) III (RRID:CGC_VC691); Pin1-like KO: pinn-1(tm2235) II (this 364 bp deletion runs the ORF into a stop codon after 8 codons). Most crosses were followed by PCR analysis to detect deletions, and by monitoring nuIs5’s GFP expression in glr-1-expressing interneurons using a high power fluorescence dissecting scope. dapk-1 over-expressing construct juEx1933 was followed by its coinjection marker, which produces RFP expression on AIY neurons. The unc-51 mutation was followed by uncoordinated phenotype and sequencing. DsRed::LGG-1 was followed by DsRed expression in cells throughout the body and by GFP expression in the pharynx (from the myo-2 promoter). We constructed the following strains: dapk-1 in excitotoxicity (by crossing VC432 and ZB1102) IMN26: dapk-1(gk219) I; glt-3(bz34) IV; nuIs5 V; DAPK-1 overexpression in excitotoxicity (by crossing ZB1102 and CZ9277) IMN25: glt-3(bz34) IV; nuIs5 V; juEx1933 [P hsp16 ::DAPK-1; P ttx-3 ::RFP]; Autophagy suppression in excitotoxicity (by crossing ZB1102 and CB369) IMN22: glt-3(bz34) IV; nuIs5 , unc-51(e369) V; Autophagy reporter in excitotoxicity IMN24: glt-3(bz34) IV; nuIs5 V; Ex[P lgg-1 :: DsRed:: LGG-1; P myo-2 :: GFP]; CaMKK deletion in excitotoxicity: ckk-1 (ok1033) III; glt-3(bz34) IV; nuIs5 V. DANGER-like deletion in excitotoxicity: (by crossing EM128 and ZB1102): mab-21(bx53) III; glt-3(bz34) IV; nuIs5 V; Pin1-like deletion in excitotoxicity: IMN30: pinn-1(tm2235) II; glt-3(bz34) IV ; nuIs5 V (± dapk-1(gk219) I).
The level of necrotic neurodegeneration in head neurons was monitored using an inverted scope and Nomarski Differential Interference Contrast (DIC). Animals with no anesthetics were examined on a fresh chunk of agar of nematode culture flipped upside down over a slide. Swollen “vacuolated-looking” cells located in the area of the nerve ring were counted as head neurons undergoing necrosis, as described previously [37,41]. Briefly, we use freshly growing mixed-stage animals, and we record the developmental stage and the number of “vacuolated” dying neurons of each animal that we see while scanning through the agar chunk, thus creating a “snap-shot” of the transitory number of dying neurons exhibited by the population of animals at the time of analysis. As mentioned above, in the excitotoxicity strain the number of “vacuolated” cells observed at each developmental stage typically goes up until L3 (with the maturation of Glu signaling in the worm), and then declines with the engulfment of cell copses. This analysis is repeated several times, with several isolates of each strain. For confirmation of suspected effects in new mutant combinations, a representative part of the analysis is done blindly by independent observers. Similar numbers of control and test animals are recorded in each session. Records from all these sessions is pooled together to calculate average neurodegeneration from large number of animals in each strain and each stage (50–200 in each data bar). We occasionally confirmed these dying neurons as neurons postsynaptic to Glu connections by verifying their labeling with the GFP co-expressed in nuIs5 animals under the glr-1 promoter. For heat-shock-induced overexpression of DAPK-1, animals carrying [P hsp16 ::DAPK-1; P ttx-3 ::RFP] were placed in a 35°C incubator for 2 hours to induce activation of heat shock promoter, left to rest for about 30 minutes and scored regularly for two days for the extent of swollen degenerating neurons (protocol & strains coordinated with the Chisholm group, UCSD ). Experimental animals (animals with extrachromosomal array) and control animals (animals lacking extrachromosomal array) were obtained, identified and scored on the same day from the same pool of heat-shocked animals. For epistasis analysis between given excitotoxicity-modifying mutations X and Y, we calculated the effect of each mutation for each developmental stage as: Fold effect of X = (the average number of dying head neuron in the excitotoxicity strain in the presence of mutation X)/(the average number of dying head neuron in the starting excitotoxicity strain), with a similar calculation for mutation Y. The expected cumulative fold effect of mutations X and Y when working independently in parallel pathways is (fold effect of X) * (fold effect of Y). The calculated expected number of dying neurons presented on the graphs in Figures 4 and 6 is (the average number of dying head neuron in the starting excitotoxicity strain) * (fold effect of X) * (fold effect of Y). Error bars represent SE. Statistical significance of difference between control groups and experimental groups was analyzed using z-test score (as the significance of these differences is calculated for large populations of ~100 animals in each group).
Fluorescence microscopy analysis of autophagy
Animals carrying Ex [P lgg-1 :: DsRed:: LGG-1; P myo-2 :: GFP]; glt-3(bz34) IV; nuIs5 V and Ex [P lgg-1 :: DsRed:: LGG-1; P myo-2 :: GFP]; nuIs5 V were analyzed for the extent to which glr-1 expressing command interneuron (labeled with GFP) were undergoing autophagy (i.e., show DsRed punca or increased DsRed intensity). Animals were mounted on a 2% agar pad. To compare average DsRed intensity between worms we identified the cell with the highest DsRed intensity as 100% and then compared the other neurons to this cell (following the procedure used by the Tavernarakis lab, who developed this DsRed::LGG-1 marker ). A few cells were analyzed in each animal and the average intensity from a total from 20–25 animals was calculated. We also counted the average of the number of green-labeled neurons showing DsRed puncta as another indication of neurons undergoing autophagy and compared control (nuIs5 only) versus experimental (glt-3; nuIs5) groups.
Blockade of autophagy with 3MA
For 3-methyladenine (3MA) treatment, mixed stage animals from the excitotoxicity strain (glt-3; nuIs5) or the DAPK-inhibited excitotoxicity strain (dapk-1; glt-3; nuIs5) were incubated overnight with 10 mM 3-MA (Sigma, M9281) dissolved in 1% DMSO (experimental group) or 1% DMSO alone (control). Plates were supplemented with E. coli OP50. The extent of neurodegeneration was recorded 20–24 h after treatment.
Behavioral assays on nematode locomotion and worm paralysis were performed blindly following standard methods of behavioral analysis. For duration of Glu-regulated spontaneous forward mobility we followed the protocol of the Maricq group (U Utah) . We did not measure mobility in strains that show excitotoxic neurodegeneration because the degeneration or rescue of command interneurons can change the results of this assay. For aldicarb assays we followed the Nonet lab protocol (Washington U) . Briefly, we soaked worm plates with aldicarb to a final concentration of 0.5 mM and added food. We placed ~30 freshly growing young adult animal onto these plates. The percentages of paralyzed worms were recorded every 15 minutes for a period of 2 hours.
We thank Dr. Monica Driscoll, Dr. Christine Li, and all members of the Mano & Li labs for their advice and support. We thank Drs. Andrew Chisholm, Andres Villu Maricq, Jeremy Dittman, King-Lau Chow, Nektarios Tavernarakis, and Alicia Melendez for reagents & advice on associated assays, Dr. S. Mitani (Tokyo Women’s Medical University School of Medicine), the C. elegans Gene Knockout Consortium, and the C. elegans Genetic Center for providing deletion alleles.
This project was supported by the Sinsheimer Foundation (P60134007) and the American Heart Association, National Center (0635367 N) to I.M. The Mano lab is also supported by the Research Centers in Minority Institutions (RCMI) grants 5G12RR003060-26 & 8G12MD7603-28 and by the National Science Foundation (IOS 1022281). J.D.R. was supported by NIH’s RISE program at CCNY. Strain collections facilities are funded by: CGC - NIH Office of Research Infrastructure Programs (P40 OD010440); NBRP - Ministry of Education, Culture, Science, Sports and Technology, Japan.
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