Glial loss of the metallo β-lactamase domain containing protein, SWIP-10, induces age- and glutamate-signaling dependent, dopamine neuron degeneration

Across phylogeny, glutamate (Glu) signaling plays a critical role in regulating neural excitability, thus supporting many complex behaviors. Perturbed synaptic and extrasynaptic Glu homeostasis in the human brain has been implicated in multiple neuropsychiatric and neurodegenerative disorders including Parkinson’s disease, where theories suggest that excitotoxic insults may accelerate a naturally occurring process of dopamine (DA) neuron degeneration. In C. elegans, mutation of the glial expressed gene, swip-10, results in Glu-dependent DA neuron hyperexcitation that leads to elevated DA release, triggering DA signaling-dependent motor paralysis. Here, we demonstrate that swip-10 mutations induce premature and progressive DA neuron degeneration, with light and electron microscopy studies demonstrating the presence of dystrophic dendritic processes, as well as shrunken and/or missing cell soma. As with paralysis, DA neuron degeneration in swip-10 mutants is rescued by glial-specific, but not DA neuron-specific expression of wildtype swip-10, consistent with a cell non-autonomous mechanism. Genetic studies implicate the vesicular Glu transporter VGLU-3 and the cystine/Glu exchanger homolog AAT-1 as potential sources of Glu signaling supporting DA neuron degeneration. Degeneration can be significantly suppressed by mutations in the Ca2+ permeable Glu receptors, nmr-2 and glr-1, in genes that support intracellular Ca2+ signaling and Ca2+-dependent proteolysis, as well as genes involved in apoptotic cell death. Our studies suggest that Glu stimulation of nematode DA neurons in early larval stages, without the protective actions of SWIP-10, contributes to insults that ultimately drive DA neuron degeneration. The swip-10 model may provide an efficient platform for the identification of molecular mechanisms that enhance risk for Parkinson’s disease and/or the identification of agents that can limit neurodegenerative disease progression.

Although cell autonomous mechanisms remain a focal point for many investigations seeking insights into determinants of neurodegeneration, increasing attention has been given to astrocytic mechanisms that can sustain neuronal viability, in the context of constant Glu stimulation that could otherwise lead to cell death. These mechanisms include the shuttling of metabolic intermediates such as lactate to neurons that can help sustain ATP synthesis [25][26][27], the buffering of extracellular ions such as K + , since excess extracellular K + due to chronic ion channel activation and Na + /K + ATPase dysregulation can contribute to excess neuronal activation [26,28,29], and the efficient clearance of extracellular Glu that both limits the amplitude of synaptic and extrasynaptic Glu signaling but also Glu-driven neuronal degeneration [26,30,31]. Astrocytic Glu clearance is mediated by multiple Na + -dependent Glu-transporters of the SLC1 family (e.g. GLT1/rodents, EAAT2/humans) that terminate Glu signaling via binding and uptake of Glu in proximity to synaptic release sites [13,31,32]. A second astrocytic Glu transporter that participates in extracellular Glu homeostasis is xCT (SLC7A11), the transporter subunit of a dimer that supports intracellular Glu exchange for extracellular cystine. xCT is generally thought to act oppositely to SLC1 transporters, balancing control of extrasynaptic Glu levels with the provision of precursor (cysteine) for astrocytic glutathione synthesis [33][34][35][36].
Due to their significant impact on synaptic and extrasynaptic Glu homeostasis, Glu transporters and exchangers have been widely studied to determine their contribution to Gluinduced neural degeneration as well as in efforts to manipulate their activity and expression for therapeutic ends [36][37][38]. For example, Rothstein and coworkers identified β-lactam antibiotics, typified by the cephalosporin-type agent ceftriaxone (Cef), as capable of elevating GLT1 expression in vitro and in vivo, protecting neurons from Glu toxicity, and enhancing longevity in an ALS mouse model [13]. Subsequently, many investigators have demonstrated the neuroprotective activity of Cef administration in rodents [39][40][41], with evidence supporting antibiotic modulation of both GLT1 and xCT expression [13,36,42], although candidates targeted by the antibiotic in glia to induce transporter expression have, until recently, been unidentified.
In a screen for novel genes that control DA signaling in the nematode, C. elegans [43], we identified a glial-expressed gene, swip-10, whose mutation induces hyper-excitability of DA neurons and elevates rates of vesicular DA release, culminating in the hyperdopaminergic phenotype, Swimming induced paralysis (Swip) [44]. These studies also demonstrated a critical role for Glu signaling in establishing the paralytic phenotype of swip-10 mutants [44]. Swip-10 is conserved across phylogeny, with the unstudied gene, Mblac1, as the putative mammalian ortholog. Both SWIP-10 and MBLAC1 proteins are metallo β-lactamase domain (MBD)-containing proteins [44], with residues key for metal binding and catalysis conserved across worm and vertebrate proteins. Although the substrate hydrolyzed by SWIP-10/MBLAC1 enzymatic activity is currently unknown, we recently established that MBLAC1 is a specific, high-affinity target for Cef [45]. We presented evidence that Cef binding activity in brain lysates could be totally eliminated by MBLAC1 immunodepletion therefore supporting the hypothesis that MBLAC1 may be the exclusive, non-microbial target of Cef in vivo. These findings also suggest that further study of SWIP-10/MBLAC1 may reveal mechanisms normally engaged to protect neurons from chronically elevated extracellular Glu and a path to the identification of novel neuroprotective agents. A key piece of data lacking in this hypothesis, however, is evidence that loss of SWIP-10/MBLAC1 either induces Glu-dependent neural degeneration or eliminates the neuroprotective actions of Cef.
Here, we capitalize on the ease of monitoring the morphology and degeneration of C. elegans DA neurons engineered to stably express green fluorescent protein (GFP) to examine a requirement for swip-10 expression in limiting Glu-dependent DA neuron degeneration. We find that swip-10 mutants demonstrate a striking, progressive degeneration of DA neurons that can be suppressed by glial expression of wild type swip-10 and by mutation of Ca 2+ permeable Glu receptor mutants. Through our studies, we provide evidence that a cell non-autonomous action of SWIP-10 sustains DA neuron viability in the context of excess Glu signaling and elevations of cytosolic Ca 2+ that we hypothesize leads to increased cellular stress and, ultimately, apoptotic cell death. Our findings support SWIP-10 (and by extension MBLAC1) as a key protective agent whose further study may yield important insights into risk factors for progressive neurodegenerative disorders and their treatment.

Dopamine neuron degeneration observed in loss of function swip-10 alleles
Given the Glu signaling-dependent, Swimming-induced paralysis (Swip) phenotype present in swip-10 mutants [44], and evidence from the latter study that swip-10 DA neurons are hyperexcitable, as assessed by a cytoplasmic Ca 2+ reporter (GCamp), we sought to determine whether these animals might display signs of excitotoxic neural degeneration. We examined the DA neurons of multiple mutant swip-10 alleles crossed to BY250, a strain that stably expresses the integrated transcriptional fusion p dat-1 ::GFP (vtIs7) (Fig 1A) [46]. We focused our evaluations on CEP DA neurons, and quantitatively evaluated degeneration by three distinct morphological assessments: 1) neurite truncations and breaks in GFP-labeled dendrites ( Fig  1B and 1C), 2) shrunken cell soma ( Fig 1D) and 3) missing cell soma (Fig 1E), as previously described [47,48]. From these categories, we also calculated an overall degeneration score where the appearance of any of the components qualifies an animal as displaying CEP degeneration [48]. We found that all three available swip-10 alleles (vt29 and vt33 from our forward genetic screen, and the larger deletion allele, tm5915) exhibited elevations in the degeneration index, relative to wildtype animals (Fig 1F-1I). To further support that mutation of swip-10 induces morphological changes in DA neurons, versus a sequestration or inactivation of cytoplasmic GFP, we corroborated our findings using a DA neuron-targeted, membrane-bound reporter (p dat-1 ::myrRFP) which also yielded evidence of tm5915 DA neurodegeneration (S1 Fig). Interestingly, evaluation of swip-10 impact on C. elegans glia broadly (marked by the ptr-10 promoter driven myrRFP) or on CEPsh glia that ensheath CEP DA neurons specifically These findings suggest that swip-10 mutation induces a localized, cell non-autonomous effect on the integrity of neighboring DA neurons.

Electron microscopy confirms DA neuron degeneration in swip-10 mutants
To be sure that our fluorescent reporters of DA neuron morphology were faithfully reporting structural changes in DA neurons, we assessed CEP cilia of swip-10 via electron microscopy (EM). Previously, we used this approach to document damage to CEP dendrites in the context of 6-OHDA induced DA neuron degeneration [49]. The tm5915 deletion allele was selected for EM studies of swip-10 induced neural degeneration, though as noted above, all mutants demonstrated comparable degeneration. The morphology of CEP neuronal processes is well characterized at the ultrastructural level [50], especially the specialized cilium at the tip of the CEP dendrite, which can be visualized in transverse thin sections through the lips of adult C. elegans (Fig 2A) [51, 52]. Using relative position and the defined morphological characteristics of CEP DA neurons, such as the electron dense cuticular branch or nubbin associated with their cilia to anchor the dendrite to the cuticle [52] and the presence of the electron dense clumps of tubule-associated material (TAM) previously shown to be characteristic of CEP cilium [51], we were able to identify multiple anomalies in tm5915 CEP structure. These defects include changes in the size and appearance of the nubbin (Fig 2B), loss or misplacement of TAM and microtubules ( Fig 2C and 2D), and the presence of large or small vacuoles in several locations either below or above the axoneme (Fig 2E and 2F). A summary of the swip-10 mutant CEP cilium defects is depicted in Fig 2G. In addition to the defects described above, half of the CEP dendrites of swip-10 mutants were missing any cilium that extended beyond the axoneme. These TEM studies confirm that swip-10 mutation results in striking DA neuron morphological changes.

Dopamine neuron degeneration increases with age
In order to determine whether the DA neuron degeneration observed in swip-10 animals represents a late onset phenomenon and/or might arise from a progressive perturbation across development, we assayed DA neuron degeneration in swip-10 mutants across various postembryonic ages. We observed that tm5915 animals display time-dependent indications of DA neuron degeneration that are distinct from the changes seen with wildtype animals (Fig 3D). In wildtype animals, signs of DA neuron degeneration are evident only in older, adult animals whereas signs of degeneration are already evident in tm5915 animals by day 1 (L1 stage) of larval development (Fig 3D). A breakdown of the components that comprise the overall degeneration score of tm5915 mutants is revealing, where non-uniform patterns are evident across measures. Although we were unable to follow individual DA neuron morphological changes over time, our population findings are suggestive of a progressive form of degeneration at the single neuron level, with dendritic breaks and truncations as earliest signs of degeneration ( Fig  3A), followed by the appearance of shrunken soma (Fig 3B), and then by missing soma ( Fig  3C). Overall similar patterns are evident with wildtype animals, just appearing much later in life. Together our findings indicate that swip-10 mutation begins to disrupt the health of DA neurons early in development with the appearance of indices of morphological perturbations arising in distal processes that progress to neuronal death.

Mutation of glial-expressed swip-10 drives DA neuron degeneration
Although the Swip behavior of swip-10 mutants at the L4 stage arises as a consequence of excess DA signaling [43], this paralysis is a cell non-autonomous consequence of glial, and not DA neuron, expression of swip- 10 [44]. To determine whether the degeneration of DA neurons is similarly a consequence of mutation of glial swip-10, we expressed a full length wild type swip-10 cDNA fused to GFP (swip-10::GFP) under control of glial and DA neuron promotors. Fig 4A demonstrates that pan-glial swip-10 expression, as achieved through use of the ptr-10 promoter [53], robustly rescues DA neuron degeneration of tm5915 animals, comparable to that achieved with a genomic construct that encodes swip-10 and the upstream elements needed to achieve full rescue of Swip [44]. Significant rescue of DA neural degeneration was also achieved with the CEP sheath glia-specific promotor hlh-17 [53]. In contrast, DA neuron specific expression of swip-10, driving expression with the dat-1 promoter, failed to restore normal morphology. Together, these findings support the conclusion that glial expression of swip-10 is required to maintain the normal viability of DA neurons.

swip-10 support for neuronal health may be related to glial ensheathment
Although not explored extensively, we sought to understand whether neural degeneration in swip-10 mutant animals is limited to the DA neurons. We chose to evaluate swip-10 mutant (tm5915) animals bearing reporters to demarcate OLL and BAG neurons. Glutamatergic OLL neurons are similar in location and morphology to dopaminergic CEP neurons, are mechanosensitive like CEP neurons, and share an association with glia (OLLsh) that ensheath OLL processes. Carbon-dioxide sensing, glutamatergic BAG neurons are similar in location and morphology to the CEP neurons, although not associated with direct ensheathing or socket glia. We observed degeneration of glutamatergic OLL neurons ( Fig 4B) but not of BAG neurons ( Fig 4C). These findings, along with rescue of DA neurodegeneration through glial reexpression of swip-10, reinforce a key role for glia in maintaining the viability of C. elegans DA neurons.  (A) swip-10(tm5915) mutants display dendritic breaks/truncations early in development, increasing in frequency with age. (B) swip-10(tm5915) mutants display shrunken soma earlier in development, increasing in frequency with age compared to N2. (C) swip-10(tm5915) mutants are missing soma earlier in development, increasing in frequency with age compared to N2. (D) As assessed by the combined degeneration index, both N2 and swip-10 DA neuron degeneration increases with age, though the swip-10 mutant DA neurons display degeneration at earlier ages than N2. Analyzed by two-way ANOVA with significant differences by age ( ÃÃÃÃ P<0.0001) and by genotype ( ÃÃÃÃ P<0.0001) and significantly different age by genotype interaction ( ÃÃÃÃ P<0.0001).

Glu signaling contributes to DA neuron degeneration induced by swip-10 mutation
Error bars represent ± SEM, with n = 105-150 animals per strain per stage. https://doi.org/10.1371/journal.pgen.1007269.g003 Neural degeneration, more generally, can be triggered by extrinsic or intrinsic activation of cell death genetic programs, first elucidated at a molecular level in C. elegans [59][60][61][62]. Additionally, disruptions of vital cellular processes (e.g. ATP production, membrane permeability, ion gradients or cytoskeletal organization) by genetically encoded neurotoxins or following exposure to reactive chemical species [63][64][65] have been shown to lead to the death of neurons. Lastly, excitotoxicity, a form of neurodegeneration with features of both apoptotic and necrotic cell death, is well known in mammalian brain preparations and typically observed in the context of over stimulation of Glu-responsive, ionotropic receptors [10,65,66]. Our prior findings that DA neurons in swip-10 animals display elevated excitability that is dependent on Glu signaling [44] encouraged our consideration of the latter mechanism of DA neuron degeneration. We therefore quantified DA morphological changes in swip-10 animals bearing loss of function mutations in genes supporting synaptic Glu packaging and Glu signaling, as well as mutations in genes encoding transporters thought to modulate extracellular Glu levels. First, we examined contributions of vesicular Glu transporters (vGLUTs). These proteins are responsible for packaging Glu into synaptic vesicles prior to release [67,68]. There are three genes that encode proteins homologous to VGLUTs in C. elegans (eat-4, vglu-2 and vglu-3) [69][70][71] with eat-4 being the only one functionally characterized to date [68,72]. Loss of individual vGLUTs (Fig 5A) had no effect on DA neuron morphology. Interestingly, whereas eat-4 mutation significantly reduced the Swip behavior of swip-10 mutants [44], the same eat-4 allele failed to blunt the degeneration of DA neurons in tm5915 animals. vglu-2 mutation was also unable to reduce DA neuron degeneration. In contrast, loss of vglu-3 significantly, suppressed DA neuron degeneration (Fig 5A), suggesting a contribution of vesicular Glu signaling, directly or indirectly, to swip-10 DA neuron degeneration.
Mammalian glia express multiple Na + -dependent Glu transporters (GLTs) of the SLC1 family that support efficient clearance of Glu after release at synapses and their dysfunction figures prominently in investigations of Glu-dependent neuronal injury and death [31]. Additionally, our previous studies [44] demonstrated that mutation of several GLTs (glt1, glt3 and glt4) conferred DA-dependent Swip. However, we found that mutation of individual glt genes failed to induce DA neuron degeneration ( Fig 5B).
A second, glial Glu transport system, xCT, regulates extra-synaptic Glu levels, acting as a cystine/Glu exchanger [36]. xCT imports extracellular cystine in exchange for intracellular Glu, and thus altering the expression or activity of this transporter can modulate extracellular Glu levels. xCT is a member of the mammalian heteromeric amino acid transporter (HAT) family, for which there are 9 C. elegans homologs, with the highest homology for xCT being to AAT-1 and AAT-3 [73]. To determine whether xCT-like proteins could contribute to DA neuron degeneration, we generated tm5915 double mutants with all available aat mutants. Of the 7 xCT homologs tested, we found that loss of aat-1 uniquely attenuated the DA neuron degeneration of tm5915 ( Fig 5C). These findings implicate non-vesicular Glu release as a contributor to swip-10 DA neuron degeneration. To determine if both vesicular Glu release supported by VGLU-3 and transporter-mediated Glu release supported by AAT-1 act in parallel or via a shared pathway to support DA neuron degeneration, we examined DA neuron morphology in an aat-1;vglu-3 double mutant. We found no enhancement of the suppression of the tm5915 degeneration beyond that of the individual mutants (S4 Fig). These findings are consistent with common mechanisms, downstream of extracellular Glu availability through either vesicular or non-vesicular Glu secretion mechanisms, as determinant of the quantitative extent of swip-10 DA neuron degeneration.
Post-synaptically in both vertebrates and nematodes, Glu binds and activates ionotropic and metabotropic receptors (iGluRs and mGluRs, respectively) [74,75]. To further pursue the hypothesis that mutation of swip-10 triggers DA neuron degeneration via excess Glu signaling, we examined DA neuron morphology in tm5915 lines bearing available mutant alleles for the iGluRs and mGluRs. Among the twelve GluR mutants tested, we found that loss of either the NMDA-type iGluR, nmr-2, or loss of the AMPA-type iGluR, glr-1 [76], significantly suppressed swip-10 DA neuron degeneration ( Fig 6A). Interestingly, these GluRs are distinct from the GluRs previously shown to suppress the paralysis phenotype of swip-10 mutants (glr-4, glr-6 and mgl-1) [44]. A double mutant of glr-1 and nmr-2 did not further suppress tm5915 degeneration beyond that seen with either mutant alone, suggesting that these receptors support neurodegeneration through a common pathway (Fig 6B). To further substantiate that excess GluR signaling via NMR-2 and GLR-1 could support our swip-10 observations, we selectively overexpressed these receptors in DA neurons and examined CEP neuron morphology. As hypothesized, we detected statistically significant DA neuron degeneration, as compared to non-transgenic lines, similar to that observed in swip-10 mutants (Fig 6C).

Contributions of changes in intracellular Ca 2+ to swip-10 induced DA neuron degeneration
The evidence presented above of a role of Ca 2+ -permeant iGluRs [77] in DA neuron degeneration, as well as our prior findings that swip-10 DA neurons demonstrate an exaggerated Ca 2+ elevation in response to food contact [44], suggested to us that DA neuron degeneration in these animals could reflect activation of Ca 2+ -dependent programs linked to apoptotic and/or necrotic cell death [78][79][80]. Consistent with this idea, we found that loss of the primary endoplasmic reticulum (ER) Ca 2+ storage/binding protein, calreticulin (crt-1) protected against swip-10 DA neuron degeneration (Fig 7A). Excessive activation of the Ca 2+ -activated protease calpain-1, has been shown to lead to cellular damage, including neurodegeneration, in both mammals and C. elegans [81][82][83]. In keeping with these findings, a loss of function mutation of clp-1, the C. elegans calpain-1 ortholog, significantly attenuated the DA neuron degeneration of tm5915 animals (Fig 7A). Together, these results support the hypothesis that inappropriate or excessive elevations of intracellular Ca 2+ support swip-10 DA neurodegeneration. In mammals, aberrant excitotoxic Ca 2+ signaling can generate reactive oxygen species (ROS) leading to activation of cell stress pathways that drive neuronal cell death [84,85]. To explore this idea, we inspected swip-10 animals for signs of oxidative stress by monitoring reporter expression from P gst-4 ::GFP. The gst-4 gene encodes a glutathione-s-transferase, and is a target for the ROS responsive transcriptional regulator SKN-1(C. elegans Nrf2 ortholog) [86,87]. As shown in Fig  7B and 7C, tm5915 animals demonstrate a significant elevation in P gst-4 ::GFP expression. As a measure of ER stress, we monitored the transcriptional reporter, P hsp-4 :GFP [88,89]. Although tm5915 animals did not show indications of basal ER stress with this marker (Fig 7D and 7E), they were more sensitive to the pharmacological ER stressor, tunicamycin, compared to N2 animals (Fig 7D and 7E).

Evidence of an apoptotic pathway in swip-10 DA neuron degeneration
Glu-induced excitotoxicity has been reported to arise from multiple mechanisms, including necrosis, autophagy, and apoptosis [90], processes that also contribute to cell death in the nematode [61]. Cells dying by necrosis exhibit cell swelling and vacuolization [61], which we do not observe in swip-10 animals (S5 Fig). In contrast, as described in Fig 1, DA neurons in swip-10 mutants display blebbing or breaks in processes (Fig 1C and 1F) and shrunken soma ( Fig  1D and 1G), features characteristic of apoptosis [91]. Consistent with this idea, we found that gain of function ced-9 mutant animals [92] and loss of function ced-4 and ced-3 mutants, wellknown contributors to programmed cell death [59], significantly suppressed tm5915 DA neuron degeneration (Fig 8A). Apoptosis in the context of normal developmental programmed cell death is tightly coupled to cell corpse engulfment [93], with two partially-redundant and parallel pathways involving ced-1/ced-6 [94,95] and ced-10 [96] responsible for recognition of dying cells and initiation of cell corpse clearance. Little is known concerning the integration of death and engulfment programs in relation to DA neuron cell death, though Offenburger recently reported contributions from both ced-6 and ced-10 linked engulfment mechanisms in 6-OHDA induced DA neuron degeneration [97]. In contrast, we found that genetic disruption of individual genes associated with ced-1/ced-6 and ced-10 had no effect on measures of swip-10 DA neuron degeneration (Fig 8B). These findings suggest that swip-10 DA neuron degeneration arises from the activation of a cell-autonomous apoptotic pathway, one that draws little observable support from known engulfment mechanisms.

Discussion
Overall, our findings reveal that loss of glial-expressed swip-10 results in DA neuron degeneration through a process supported by excess Glu signaling through Ca 2+ -permeant ionotropic Glu receptors and Ca 2+ -dependent cell death mechanisms that engage apoptotic cell death pathways, as summarized in Fig 9. Although, we predominantly characterized swip-10 DA neuron viability in gravid (egg-laying) adults, time-dependence studies indicate that degeneration is evident by day 2 post-hatching, and increases on all degeneration measures across the lifespan. A predominant display of fragmented or truncated dendrites in young animals versus  shrunken or lost soma at later stages (Fig 3) suggests that degeneration in individual neurons is progressive, first emerging as altered neurite structure, followed by engagement of all compartments and eventually resulting in disappearance of some DA neurons altogether. This progressive pattern of degeneration is commonly seen with neurons suffering from energy depletion, that can be triggered by excessive stimulation or through metabolic poisoning [98,99].
Most of our observations were obtained with a DA neuron-specific, cytosolic, fluorescent reporter, findings corroborated using a membrane anchored reporter (Figs 1 and S1). Subsequent studies of swip-10 mutants using electron microscopy to image DA dendrites and cilia provided clear evidence of physical alterations (Fig 2) that we believe reflect the declining health of the DA neurons, versus a direct action of swip-10 or its immediate effectors though further studies are needed. Additional EM studies would also be valuable in investigating the degeneration of swip-10 mutants at the level of the DA neuron cell bodies, axons, and synapses. There are significant technical challenges associated with identifying these cells and processes in the densely populated nerve ring, though future studies that make use of correlated light electron microscopy (CLEM) can be envisaged [100].
The discovery that DA neurons degenerate in swip-10 animals was initially surprising as our identification of swip-10 derives from a hyperdopaminergic behavioral phenotype [44], though we previously demonstrated reduced DA levels in these animals [43]. Since swip-10 DA neurons exhibit increased excitability and tonically-elevated DA secretion rates [44], we hypothesize that the degenerative process we have characterized likely contributes to a perturbation of mechanisms that insure a tight control over DA release (e.g. DA autoreceptors), along with a diminished capacity for DA clearance, leading to Swip. Alternatively, a degeneration-induced loss of DA signaling capacity could result in a hypersensitivity of postsynaptic swip-10 animals significantly respond to the ER stressor, tunicamycin, with swip-10 mutants significantly more sensitive to tunicamycin. Analyzed by one-way ANOVA and Sidak's post-tests (A and E), or analyzed by Student's t test (C), ÃÃ , ÃÃÃ , and ÃÃÃÃ indicates a P<0.01 <0.001, and <0.0001 respectively, ns = nonsignificant (P>.05), error bars represent ± SEM, with n = 105-150 animals per strain.
https://doi.org/10.1371/journal.pgen.1007269.g007 Having generated evidence for an age-dependent degenerative process impacting the morphology of swip-10 DA neurons, we pursued mechanistic studies through a combination of genetic and imaging techniques. Such approaches have provided for a systematic elucidation of mechanisms underlying both programmed and environmentally-triggered cell death [59, [101][102][103][104]. In addition to the apoptotic pathways that drive programmed cell death during SWIP-10 and dopamine neuron degeneration development, molecular determinants of later stage necrotic neuronal death, that arise as a result of the constitutive activity of mutant ion channels [105,106] and ligand-gated Glu receptors [107], have been investigated. A potential role for excess Glu signaling in swip-10 DA neuron degeneration seemed plausible given the contribution of Glu receptors and Glu transporters to Swip reported in our prior study [44]. In this regard, the groups of Driscoll and Mano have provided evidence that necrotic cell death arises with excess Glu signaling that occurs from a combined loss of Glu clearance and a hyperactive, constitutively active form of the alpha subunit of the G-protein, G s [108][109][110]. Although swip-10 DA neuron death shares features associated with the degeneration described in Mano's studies, specifically the contributions from the iGluR, glr-1 (Fig 6A), and the intracellular Ca 2+ sequestering protein, crt-1 (Fig 7A), our analysis also reveals a number of differences. Thus, besides a lack of morphological evidence of swollen, vacuolated soma seen in prior studies (S5 Fig), we found no evidence for a contribution to DA neuron degeneration of the adenyl cyclase ortholog, acy-1, nor could we implicate the autophagy-associated, cell death protein kinase, dapk-1 (S6 Fig) [108, 109]. We also obtained evidence that the damaging effects of swip-10 mutation are quite distinct from those observed with 6-OHDA induced DA neuron cell death. For example, the degeneration of DA neurons that arises within a day following 6-OHDA administration to wildtype worms lacks contributions from genes that participate in programmed cell death mechanisms [49], whereas, as we discuss below, contributions of these genes are evident in the swip-10 model. Moreover, recent studies indicate that ced-6 and ced-10 dependent engulfment pathways support 6-OHDA induced loss of DA neurons [97], whereas we found no contribution of these engulfment genes to swip-10 effects (Fig 8B). Moreover, Offenburger and colleagues have reported that 6-OHDA induced DA neuron death is exacerbated by mutation of the Ca 2+ chaperone crt-1 whereas we demonstrated crt-1 mutation confers neuroprotection [111]. Together, these findings indicate that the DA neuron degeneration induced by swip-10 mutation is an altogether unique form of neural degeneration as compared to prior glutamatergic and exogenous neurotoxin models.
A critical step in defining the mechanism associated with swip-10 DA neuron degeneration is to determine the site(s) where wildtype swip-10 expression is required to support normal DA neuron morphology. As with the rescue of DA-dependent Swip behavior [44], we found that glial swip-10 re-expression, both genomic and cDNA, rescued swip-10 DA neuron degeneration, whereas DA neuron expression of swip-10 was without effect. These findings attest to a cell non-autonomous mode of action and raise the possibility that glial loss of swip-10 may damage the glial cells themselves, rendering them unable to engage in secretory or contactmediated support for DA neurons. Although non-quantitative, we detected no obvious morphological differences between wildtype and swip-10 glia (S2 Fig), which may indicate that swip-10 expressing glia are deficient in a capacity to provide specific trophic or metabolic support to DA neurons, versus participating in critical cell autonomous mechanisms. Future studies using higher resolution, EM-based methods should be pursued to refine this analysis. Importantly, we obtained rescue of swip-10 DA neuron degeneration with a promoter driving wildtype swip-10 expression in CEP sheath glia. Moreover, degeneration was apparent in OLL neurons that like CEPs are ensheathed by glia but not in BAG neurons, which lack these contacts. These studies reinforce a contribution of glia to the cell non-autonomous actions of swip-10 to sustain neuronal viability and suggest that neurons in close apposition to ensheathing glia may preferentially depend on the activity of swip-10.
Our prior studies [44] assessing Swip behavior in swip-10 mutants provided evidence of perturbed glial control of extracellular Glu that we hypothesized was responsible for the iGluR and mGluR dependence of Swip in these animals. We therefore considered the possibility that perturbed buffering of extracellular Glu by swip-10 glia also underlies DA neuron degeneration. Mammalian literature emphasizes the critical role of glial Glu buffering mechanisms as protective against Glu excitotoxicity. As first described by Olney and colleagues, Glu excitotoxicity derives from excessive synaptic Glu acting on post-synaptic iGluRs [112][113][114], a process recapitulated by the actions of iGluR agonists such as kainic acid and ibotenate [115][116][117]. Moreover, inhibition of Glu transporters and increased extracellular Glu recapitulates the pathological hallmarks of PD in animal models, including DA neuron degeneration [118]. Our findings that mutations in the Ca 2+ permeant iGluRs, glr-1 and nmr-2, protect against swip-10 DA neuron degeneration and that overexpression of these receptors leads to DA neuron degeneration in wildtype animals (Fig 6C) provide strong supportive evidence that glial mechanisms dictating the availability of extracellular Glu are likely disrupted in swip-10 animals.
Mammalian glia have been reported to modulate extracellular Glu by vesicular release [119], Glu-permeable channels [120], synaptic clearance of Glu by Na + -coupled Glu transporters (GLTs) [31], and extrasynaptic Glu buffering by the cystine/Glu exchanger (xCT) [34,35]. We found that a mutation in the vesicular Glu transporter vglu-3 attenuates swip-10 DA neuron degeneration (Fig 5A). We were surprised that an eat-4 mutation did not contribute to swip-10 induced degeneration, as such a mutation reduced Swip behavior [44]. Although the expression pattern and role for vglu-3 is undetermined, these findings raise the possibility that EAT-4 supports Glu signaling in the neural circuitry that drives DA neuron excitation in response to water, whereas VGLU-3 contributes to Glu release directly onto DA neurons, including SWIP-10 expressing glia, and drives tonic activation of Glu receptors on DA neurons and over time, excitotoxicity. Consistent with this model, distinct Glu receptors support Swip (GLR-4, GLR-6 and MGL-1) versus DA neurodegeneration (GLR-1 and NMR-2). Although we did not observe DA neurodegeneration with genetic loss of single GLT orthologs in the nematode (Fig 5B), unlike Swip [44], this may be due to genetic redundancy among the six GLTs. Indeed, studies by the Driscoll lab demonstrated that loss of one or two GLTs is insufficient to drive Glu-dependent neurodegeneration [121]. In contrast to our inability to implicate specific GLTs, we found that genetic disruption of the xCT related gene, aat-1, significantly reduced swip-10 DA neuron degeneration ( Fig 5C). As with vglu-3, the expression pattern for aat-1 in the worm is undefined, and thus additional studies are needed to determine site(s) of expression that contribute to our results. The effects of aat-1 mutation were not additive with those of vglu-3, suggesting that both genes act to support DA neurodegeneration through a common mechanism, which we propose is through the control of tonic, extracellular Glu providing tonic excitation of DA neuron expressed Glu receptors. Finally, it is important to note that mammalian xCT is upregulated by the β-lactam antibiotic ceftriaxone [36,42,122], which we have shown binds directly to the putative swip-10 ortholog MBLAC1 [45]. Moreover, research, initiated by findings of Rothstein and colleagues [13], has demonstrated that ceftriaxone is neuroprotective, including in models of DA neuron degeneration [41].
Although not exclusive, Glu-induced neural degeneration often involves activation of Ca 2+permeable NMDA type iGluRs [19,123,124] and, as noted, our studies demonstrate an important contribution of Ca 2+ -permeable C. elegans iGluRs, the NMDA-type iGluR, nmr-2, as well as the AMPA-type iGluR, glr-1 in swip-10 neural degeneration [77] (Fig 6A). Expression profiling data provides evidence that nmr-2 and glr-1 are expressed in DA neurons [125]. Since swip-10 mutant animals with loss of both nmr-2 and glr-1 do not demonstrate enhanced suppression of DA neural degeneration as compared to single receptor mutations (Fig 6B), we suggest that the flux of Ca 2+ through one of these receptors is sufficient to increase intracellular Ca 2+ sufficiently to initiate downstream signaling pathways that lead, over time, to neurodegeneration. Aberrant intracellular Ca 2+ regulation and signaling has been implicated in excitotoxic cell death [126], with evidence supporting a role for Na + /Ca 2+ -permeable degenerin/ epithelial sodium channels (DEG/ENaCs) [127][128][129], Ca 2+ -dependent proteases such as calpain [130,131], and deficiencies in ER Ca 2+ buffering [80,106] in cell death mechanisms. We found that disrupting ER Ca 2+ storage, by mutation of crt-1, or mutation of the C. elegans calpain ortholog, clp-1, significantly rescued swip-10 DA neural degeneration (Fig 7A). Ca 2+ dysregulation following excessive Glu stimulation has also been shown to engender multiple indications of cell stress including oxidative stress and ER stress [132,133], which swip-10 mutants display.
Finally, although acute Glu excitotoxicity has been more typically associated with necrosis [17, 19], evidence suggests that chronic dysregulation of Glu signaling and altered intercellular Ca 2+ homeostasis can lead to activation of apoptotic pathways [134,135], and a recent study by Anilkumar and colleagues has demonstrated that external factors, such as nutrient availability, determine whether or not excess Glu signaling triggers apoptotic or necrotic cell death (Anilkumar 2017). Consistent with this idea, genetic disruption of apoptosis in C. elegans [59] significantly reduced the DA neurodegeneration of swip-10 mutants (Fig 8A). The progressive DA neuron degeneration we detect in swip-10 animals supports the occurrence of a chronic insult and thus is in line with our genetic findings of apoptotic program engagement. However, our data suggests that swip-10 involvement of apoptotic cell death associated genes differs from the involvement of these genes in developmental programmed cell death, as loss of genes critical for cell-corpse engulfment during programmed cell death did not alter the levels of swip-10 DA neuron degeneration (Fig 8B). Although lack of a reliance on engulfment genes could be a reflection of the partial redundancy of the two major engulfment pathways, we suspect that these findings are indicative of a slower engagement of apoptotic genes in the swip-10 model. Additionally, the majority of our assays are conducted at a mid-point, with degeneration in progress, to capture various degrees of degeneration in swip-10 animals, it is possible that we have simply not assessed the correct temporal window for engulfment.
Although we present clear evidence for a significant role of excess Glu signaling in the degeneration of swip-10 DA neurons, other mechanisms besides changes in extracellular Glu homeostasis are likely to contribute to our observations since Glu homeostasis and signaling mutants afford incomplete suppression of swip-10 DA neurodegeneration. The elucidation of the normal role and genetic pathway for wildtype swip-10 in C. elegans glial cells will likely clarify other contributors to swip-10 induced neural degeneration. For example, mammalian glia have been shown to support neurons by buffering ions such as potassium (K + ) and hydrogen (H + ) [136], and by providing metabolic support via lactate, glutathione, and ATP shuttling [26]. Although only limited data speaks to glial-neuronal crosstalk in worms, we suspect that one or more of these mechanisms contribute to the diminished viability of DA neurons in swip-10 animals. As our transcriptional stress reporter data indicate a systemic increase in cellular stress mechanisms (Fig 7B-7E), it seems entirely likely that the perturbations induced by swip-10 mutation extend beyond the deficits observed in CEP (and OLL) neuron viability. Since wholesale degeneration is not evident, we suspect that the premature degeneration of DA neurons reflects a more dependent relationship of these cells on glia. The selective loss of nigrostriatal DA neurons in idiopathic PD has been suggested to derive from an intrinsic vulnerability to stress, possibly arising from the reactivity of DA itself, as well as inefficient antioxidant protection, ultimately rendering these cells more vulnerable than others to Gluinduced cell death [137]. Since genetic elimination of the capacity to synthesize DA did not reduce swip-10 DA neuron degeneration, we feel it more likely that excess Glu signaling drives degeneration in combination with a parallel loss of glial metabolic or trophic support required by DA neurons.
In summary, our findings reveal a previously unreported dependence of DA neurons on C. elegans glia, one that when disrupted leads to neuronal degeneration. DA degeneration triggered by glial loss of swip-10 appears to be progressive and dependent on excess Glu signaling through Ca 2+ permeant iGluRs. We propose that these effects lead to perturbed intracellular Ca 2+ homeostasis and, progressively, the engagement of apoptotic cell death pathways. Our work adds support to studies in mammals that indicate a critical role of proper glial function in DA neuron viability [138][139][140][141] and reveals a new worm model of Glu excitotoxicity, one likely amenable to pharmacological manipulation that could provide insights to novel therapeutics to treat human neurodegenerative disorders.

C. elegans strains
Strains were maintained as described previously [142]. We thank J. Rand (Oklahoma Medical Research Foundation); the Caenhorhabditis Genetics Center; Shohei Mitani of the National Bioresource Project at Tokyo Women's Medical University; and Shai Shaham, Niels Ringstad, and Oliver Hobert for providing the strains used in this work. N2 (Bristol) served as our wildtype strain, and unless specified otherwise, we utilized the proposed null allele, TM5915, of swip- 10 [44]. Strains used in this study are enumerated per figure appearance in S1 Table.

Plasmid construction and transgenic manipulations
In all cases, insertion of the DNA fragment of interest and the fidelity of the vector was confirmed by sequencing and all PCRs were performed using KAPA HiFi HotStart ReadyMix (Kapa Biosystems). All constructs resulted in C-terminal cDNA fusion to an unc-54 3' UTR. For the membrane bound transcriptional reporter, we used overlap PCR [143] and Gibson Assembly (NewEngland Biolabs) to subclone the 700bp dat-1 promotor into the myrRFP containing backbone from p ptr-10 :myrRFP (gift from Shai Shaham) to create pRB1349 (p dat-1 : myrRFP). For transgenic swip-10cDNA::GFP rescue experiments, DA neuron, pan-glial, and CEPsh glial expression was achieved using the previously described plasmids, pRB1157, pRB1158, and pRB1159, respectively [44]. Genomic full-length swip-10 rescue experiments were conducted as previously described [44]. For the DA neuron-specific Glu receptor experiments, a PCR product (20ng/μL) was amplified by overlap PCR [143] to include the 700bp dat-1 promoter and genomic glr-1 from the ATG start to 2890 of genomic nmr-2 from the ATG start to 2974 fused to unc-54 3' UTR for injection, along with p unc-122 :RFP (35ng/μL) and p dat-1 :myrRFP (35ng/μL).

Genetic crosses
Crosses were performed using publicly available, integrated fluorescent reporter strains to mark chromosomes in trans. Single worm PCR was performed to confirm the presence of the indicated mutation. For all deletions, we used a three primer multiplex strategy that produces PCR amplicons with a 100-200 bp difference between N2 and mutant. This method was highly effective in eliminating preferential amplification of a lower-molecular-weight species. In all cases, a synthetic heterozygous control was used to ensure that heterozygous clones could be identified. We identified recombinant lines by PCR genotyping of single worm genomic DNA lysates. All genotyping PCRs were performed with the KAPA Genotyping Kit (KAPA Biosystems). In some cases, alleles were sequenced with sequence-specific primers to verify mutation homozygosity (GeneHunter and EtonBioscience).
60x oil-immersion objective and Nikon Elements capture software. Worms were immobilized using 30mM levamisole in M9 on a fresh 2% agarose pad and cover-slipped with a 1mm cover glass before sealing with paraffin wax [144].

Neurodegeneration assay
The neurodegeneration assay was adapted from a previously described method [145]. In our case, we transferred 20 worms to normal NGM/OP50 plates as L4s and incubated these plates for 48hrs at 19˚C until animals reached the gravid adult stage, unless otherwise noted. We then picked 15 worms into 20μL of 30mM levamisole in M9 on slides prepared with a 2% agarose pad. For imaging, we utilized a Zeiss Discovery V12 inverted fluorescent microscope outfitted with a Xenon UV light source and GFP/YFP/RFP filter sets. We used a Zeiss mono FWD 16mm objective lens to visualize Green Fluorescent Protein (GFP) containing integrated transgenes, vtIs7[P dat-1 ::GFP], nsIs242[ Pgcy-33 ::GFP], wgIs328[P ser2prom3 ::GFP] selectively expressed in DA, BAG, and OLL neurons respectively, allowing us to examine neurodegeneration in a cell-specific manner. For the DA neurons, analysis was limited to CEP neurons, because out of the 8 DA neurons in C. elegans, the 4 CEP neurons display the clearest and most distinct dendritic projections and can be readily identified via both light and electron microscopy (see below). Neurons were examined for the presence of 1) breaks in the CEP dendrites 2) shrunken or 3) missing somas. Worms were counted as displaying degeneration if one or more of these features were present. Normal N2 CEP, BAG, and OLL neurons lacked any of these abnormalities at the gravid adult stage. Total animals with degeneration, shrunken and missing somas, or neurite breaks were calculated for each trial. The percentage of animals exhibiting each morphological trait was determined for graphical analysis. Animals were tested 15 animals/day on 7-9 separate days (n = 90-135 animals assayed per genotype) blinded to genotype.

Electron microscopy
N2 and swip-10 mutant animals were raised and maintained at 20˚C on E. coli OP50/NGM plates and 2-day adult animals (fixed 2 days after the L4 stage) were fixed and embedded for transmission electron microscopy (TEM) following a chemical immersion protocol [146,147]. Briefly, animals were first cut open in a cacodylate-buffered osmium tetroxide fixative, then en bloc stained in uranyl acetate, and dehydrated and embedded in Spurr resin. Thin sections were collected onto Formvar-coated slot grids and examined on a Philips CM10 electron microscope. Digital images were collected with an Olympus Morada camera on the TEM, and figures were created using Photoshop.

Fluorescence microscopy with GFP stress reporters
All fluorescent stress reporter stains were a generous gift from Dr. Matt Gill (Scripps Research Institute, Jupiter, FL). All stress reporter strains were imaged as gravid adult animals grown at 19˚C for 48hrs after transfer to a fresh OP50/NGM plate at the L4 stage. To determine levels of stress we used the transcriptional reporter strains, dvIs19 [p gst-4 :GFP] and zcIs4 [p hsp-4 :GFP] to measure oxidative stress and ER stress respectively. We adapted previously described methods [87,88]. Briefly, the overall p gst-4 :GFP fluorescence intensity/μm per 15-20 3 day adult swip-10 animals and 15-20 3 day adult N2 animals (with subtracted background fluorescence per animal) was determined, and the fold change GFP intensity compared to N2 signal was calculated for all animals assayed from one population and subsequently averaged over 4 independent days (n = 60-75 animals assayed). As a positive control for oxidative stress, we picked 15-30 L4 N2 animals to OP50 plates 2mM paraquat (Sigma) mixed with the NGM agar [148]. p hsp-4 :GFP fluorescence intensity/μm was assayed as described above. To determine susceptibility of swip-10 mutants to ER stress, we transferred 15-30 L4 N2 and swip-10 animals to NGM plates containing 10μg/mL tunicamycin (Sigma) [89]. For each of the stress reporters, images were acquired using identical imaging settings across blinded genotypes and drug treatments, via a Nikon A1R confocal microscope in the FAU Brain Institute Cell Imaging Core using a 4x objective and Nikon Elements capture and analysis software.

Statistical analyses
All statistical tests were performed and graphs generated using Prism version 7.0. Data were analyzed by Student's t-tests, one-way ANOVAs followed by Sudak or Dunnet's post-hoc tests and two-way ANOVAs, where appropriate. A P < .05 was taken as evidence of statistical significance in all cases.
Supporting information S1 Table. C. elegans strains used in the generation of data that appear in the figures.