Gasdermin-E mediates mitochondrial damage in axons and neurodegeneration

Mitochondrial dysfunction and axon loss are hallmarks of neurologic diseases. Gasdermin (GSDM) proteins are executioner pore-forming molecules that mediate cell death, yet their roles in the central nervous system (CNS) are not well understood. Here, we find that one GSDM family member, GSDME is expressed by both mouse and human neurons. GSDME plays a role in mitochondrial damage and axon loss. Mitochondrial neurotoxins induced caspase-dependent GSDME cleavage and rapid localization to mitochondria in axons, where GSDME promoted mitochondrial depolarization, trafficking defects, and neurite retraction. The frontotemporal dementia (FTD)/amyotrophic lateral sclerosis (ALS)-associated proteins TDP-43 and PR-50 induced GSDME-mediated damage to mitochondria and neurite loss. GSDME deficiency prolonged survival, ameliorated motor dysfunction, and rescued motor neuron loss in the SOD1G93A mouse model of ALS. GSDME knockdown also protected against neurite loss in ALS patient iPSC-derived motor neurons. Thus, we identify GSDME as an executioner of neuronal mitochondrial dysfunction that contributes to neurodegeneration. Graphical Abstract Highlights GSDME is expressed by neurons and activated by mitochondrial neurotoxins Activated GSDME drives axonal mitochondrial damage and neurite loss prior to cell death ALS/FTD associated TDP-43 and PR-50 induces GSDME-driven neurite loss in mouse and human iPSC-derived neurons. SOD1G93A mice show ameliorated disease progression and motor neuron loss in absence of GSDME


Highlights
• GSDME is expressed by neurons and activated by mitochondrial neurotoxins • Activated GSDME drives axonal mitochondrial damage and neurite loss prior to cell death • ALS/FTD associated TDP-43 and PR-50 induces GSDME-driven neurite loss in mouse and human iPSC-derived neurons.
• SOD1 G93A mice show ameliorated disease progression and motor neuron loss in absence of GSDME

Introduction
Neurologic diseases are often characterized by early mitochondrial dysfunction and axon loss, which precede overt cell death [1][2][3][4] . Healthy neuronal mitochondria are essential for a variety of cellular functions, including efficient ATP generation 5 , synaptic transmission 6 , ROS generation and calcium signaling [6][7][8] . Mitochondrial damage leads to release of contents including mitochondrial DNA and cytochrome-c, which drives caspase-3 activation and apoptosis 9 .
Localized caspase-3 activity can also occur in neurites and contributes to axonal damage; once a threshold is reached in the soma, caspase-3 can drive overt neuronal cell death 3 . Though the link between mitochondrial damage and caspase-3 activation is well characterized, the myriad mechanisms that amplify local mitochondrial demise in neurites, drive axonopathy and lead to neuronal dysfunction are not fully understood. Mapping the molecular mechanisms linking mitochondrial stress to axon degeneration and neuronal death may have implications for a wide range of neurologic insults.
Gasdermins have pore-forming N-terminal domains that are masked by an autoinhibitory Cterminus in the resting state. Proteolytic cleavage in the linker region between the -N and -C domains release the N-terminal fragment of GSDMs to insert and oligomerize in lipid membranes and form pores. Accumulation of GSDM pores in the plasma membrane leads to cellular swelling and overt necrosis-a process termed "pyroptosis" [10][11][12][13] . To date, most studies concerning GSDMbiology have focused on immune, epithelial or cancer cells. GSDMD, which was first identified in macrophages, is enriched in immune cells, and activated by cleaved caspase 1/11 downstream of inflammasome assembly [12][13][14] . Prior to mediating pyroptosis, N-GSDMD can also permeabilize mitochondrial membranes leading to release of cytochrome-c, mtDNA and downstream caspase-3 activation 15,16 . However, the range of cell types where GSDM family members are expressed and activated has not been well defined.
In this study, we focus on the role of one family member, GSDME, based on its expression in brain and spinal cord. Prior to its discovery as a gasdermin family member, GSDME was first named deafness associated family member 5 (DFNA5). Patients with gain of function mutations in GSDME present with progressive sensorineural hearing loss-the hair cells of the cochlea are destroyed but other tissues appear to be spared 17,18 . Early Northern blot and subsequent RT-qPCR analyses showed that DFNA5 was broadly present in mesenchymal-derived tissues, placenta, and bulk brain samples 17,19 . The molecular mechanism of GSDME activation was first elucidated in cancer cell lines, where it was shown to be cleaved by caspase-3 11 . Once released, the GSDME N-terminal fragment inserts into the plasma membrane and forms pores that drive pyroptosis 10,11 . Subsequent work has shown that GSDME can be activated in human keratinocytes 20 , the GI tract 21 , kidney 22 , mouse retinoblastoma cells 23 , and human cancer cells 11,24,25 .
The function of GSDMs in neurons and its action in axonal biology has not been studied. Here, we find that both mouse and human neurons express GSDME, and that it is enriched in neurons compared to glia. Neurons are distinct from other GSDM-positive cell types; they are non-dividing and possess long morphological processes, namely dendrites and axons. Neurons also have unique energetic demands, needing to transport mitochondria over great distances to maintain signal transduction in axons and synapses. In neurodegenerative conditions such as Parkinson's Disease (PD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS), proteins such as α-synuclein, TDP-43, SOD1 and C9ORF72-associated dipeptide repeat proteins (DPRs) disrupt mitochondrial function and induce loss of axonal processes. These changes are early steps in disease pathogenesis 1,5,[26][27][28][29] .
Our study reveals that GSDME mediates local mitochondrial damage in axons that drives neurite loss and cell death. Activated GSDME rapidly distributes to neurite-associated mitochondria to amplify membrane potential loss, slow mitochondrial trafficking, and promote neurite retraction.
In microfluidic chambers, we show that GSDME-driven mitochondrial damage can be localized to distal axons. FTD/ALS-associated proteins induced GSDME activation and localization to axonal mitochondria, driving mitochondrial depolarization and neurite loss. GSDME is activated and plays a functional role in mouse (SOD1 G93A ) and a patient-derived iPSC-motor neuron model of ALS. Genetic knockout or shRNA-based knockdown of GSDME protects against axonal damage to neurons in vitro and motor dysfunction in vivo. Thus, we identify GSDME as an important modifier of mitochondrial damage neuronal dysfunction that may play a role in neurologic disease.

GSDME is expressed in mouse and human neurons
The expression of the Gasdermin family in the central nervous system and its potential functions in neurons have not yet been well characterized. We mined publicly available single cell RNA-seq data of mouse cortex and hippocampus sorted for neurons 30,31 .The GSDM family members Gsdmd, Gsdma, and Gsdmc were largely absent, while Gsdme was expressed in both GABAergic and Glutamatergic neurons (Fig 1A) To further confirm Gsdme expression in the mouse brain, we isolated tissue samples from different brain regions of wild-type (WT) or Gsdme -/mice for transcriptional analysis, immunostaining, and immunoblotting. Gsdme mRNA was detected in cortical, cerebellar, midbrain and spinal cord lysates from WT mice, but absent in Gsdme -/-(KO) animals (Fig S1A). Anti-GSDME immunostaining of mouse brain showed a broad expression pattern in cells with neuronal morphology (Fig 1B). As expected, staining and immunoblotting of mouse brains from knockout animals showed minimal immunoreactivity (Fig S1B and Fig 1C).
To test if GSDME is enriched in neuronal cells compared to glia, we performed MACS purification of neurons vs. non-neuronal cells from mouse brain followed by western blot analysis (Fig 1D).
We found clear GSDME signal in neuronal fractions that were also positive for neuronal beta-IIItubulin (Tuj1). GSDMD, which has been mostly characterized in macrophages 14 , was absent in neurons. However, the non-neuronal fractions (Iba1+, Tuj1-) showed clear expression of GSDMD but not GSDME (Fig 1D). Consistent with this analysis, transcriptomic data 32 from FACS-isolated mouse microglia showed Gsdmd expression at baseline but sparse Gsdme detection in these same populations (Fig S1C). Co-staining sections of cortex and striatum showed high colocalization of GSDME with the neuronal marker Tuj1, and not the microglial marker Iba1 (Fig   1E), confirming neuron-specific enrichment of GSDME compared to microglia. Evidence from publicly available human single cell transcriptomic databases 30 . (Bakken et al., 2021) similarly demonstrated that GSDME, but not family members GSDMD, GSDMA or GSDMC, is expressed at baseline in human neurons (Fig S1D). We next analyzed GSDME protein expression in human brain samples. Temporal cortices from human brains were stained with anti-GSDME. We found that GSDME was expressed in multiple cortical layers; these GSDME-positive cells showed a perikaryal staining pattern consistent with cytosolic expression in neurons (Fig 1F). Taken together, this data shows that GSDME is expressed at baseline in both mouse and human neurons.

GSDME mediates neuronal cell death caused by mitochondrial toxins
Mitochondrial toxins, such as rotenone and 6-OHDA, are intrinsic apoptotic stimuli often used to induce neuronal cell death, and model features of neurodegeneration in vitro and in vivo 2,4 . In cancer cell lines, GSDME is classically cleaved and activated by caspase-3 downstream of apoptotic triggers (Fig 2A) 11 . Given that this molecule is expressed in neurons, we sought to understand if toxin treatment could activate GSDME in human and murine cells. For this study we utilized several mitochondrial toxins: rotenone and 6-OHDA (complex I inhibitor), antimycin (complex III inhibitor) and raptinal (a rapid inducer of mitochondrial damage and caspase-3). We found that treatment of primary mouse cortical neurons and human SH-5YSY cells with these mitochondrial toxins led to cleavage of full-length GSDME to the ~30 kDa N-terminal pore-forming fragment by immunoblot analysis (Fig 2B & S2A) and cell death as measured by lactate dehydrogenase (LDH) release and uptake of the membrane impermeant dye propidium iodide (PI) (Fig S1E-H). We confirmed specific GSDME cleavage following toxin treatment through immunoblot analysis of WT and GSDME KO SH-SY5Y cells generated using CRISPR-Cas9 targeting (Fig S2A). Thus, these toxins can be used as probes to explore the biological functions of neuronal GSDME.
We next determined whether loss of Gsdme impacts cell death downstream of mitochondrial toxin treatment. Gsdme KO primary mouse neurons showed significantly reduced PI or Sytox Green uptake compared to WT neurons treated with the toxins raptinal, rotenone and antimycin-A (Ant-A) (Fig 2C-G). These results were confirmed in GSDME KO SH-SY5Y cells, which were also protected from PI uptake induced by raptinal, rotenone, 6-OHDA, Ant-A and 3-NP, suggesting that GSDME mediates cell death downstream of multiple mitochondrial toxins (Fig S2B-G).
Challenge with ER stressors tunicamycin and thapsiagargin, which generate ROS and activate caspase-3, also caused a GSDME-dependent PI uptake (Fig S2I-K). These findings indicate that GSDME contributes to plasma membrane permeability both in primary mouse neurons and a human neuron-like cell line, downstream of multiple neurotoxic triggers. Toxin-induced LDH release was also partially rescued in GSDME deficient primary neurons and SH-SY5Y cells compared to wild-type controls (Fig 2H & S2H). Taken together, these experiments show that mitochondrial toxin induced necrosis (PI+ uptake and LDH release) is regulated by GSDME in human neuroblastoma cells and mouse primary cortical neurons.

GSDME activation in neurons is caspase-dependent
Given that mitochondrial toxins classically activate caspase-3, which is upstream of GSDME cleavage 10,11,20 , we next assessed whether GSDME-driven cell death is indeed caspasedependent in neurons. Treatment with the pan-caspase inhibitor zVAD-FMK blocked raptinalinduced GSDME processing into its N-terminal fragment (Fig S3A-B). Incubation of SH-SY5Y and mouse primary cortical neurons with zVAD-FMK phenocopied the effect of GSDME knockout, decreasing PI uptake after toxin treatment (Fig S3C-F). By contrast, treatment with the RIP kinase inhibitors nec-1s and GSK '872 did not reduce toxin induced PI uptake in primary neurons, suggesting that necroptosis does not participate in mitochondrial toxin-driven neuronal death (Fig   S3G-H). These data indicate that treatment of neurons with mitochondrial toxins activates a caspase-3-GSDME axis, leading to neuronal death.

Activated GSDME rapidly localizes to mitochondria
Activated (N-terminal) GSDME can bind to a range of acidic lipid residues, such as phosphatidyl serine in the plasma membrane and cardiolipin (CL) in mitochondria. 11,16,34 Following insertion into lipid bilayers, N-GSDM fragments oligomerize to form 10-21nm pores. 11,13 Neurons have large somas and fine dendritic and axonal processes (neurites). Given the unique morphology and cell biology of neurons, we hypothesized that activated GSDME may act at specific subcellular compartments to drive cellular dysfunction. To investigate the intracellular dynamics of GSDME, before and after activation, we transfected mouse cortical neurons with N-terminally tagged full-length GFP-GSDME (Fig S4A). We validated that GFP-GSDME maintained poreforming and pyroptotic ability by transfecting this construct into HEK293T cells, which lack endogenous GSDME (Fig S4B-C). When transfected cells were treated with raptinal, we observed caspase-3 activation, cleavage of GFP-GSDME, rapid PI+ uptake and pyroptotic morphology (Fig S4C-F). These changes were not observed in untransfected or GFP-transfected control cells, which lack GSDME expression (Fig S4D). Performing immunoblot analysis of the time course for GSDME activation, we observed that cleavage of both endogenous GSDME and transduced GFP-GSDME were identical and concomitant with caspase-3 cleavage in primary neurons (Fig S4G).
Following raptinal treatment of neurons, GFP-GSDME rapidly formed numerous intracellular puncta in the distal neurites by 45 minutes, and eventually proximal neurites and cell bodies by 75 minutes (Fig. 2I-J). When cortical neurons were co-transfected with a membrane-RFP marker and treated with raptinal, GFP-GSDME puncta appeared predominantly dispersed in the cytosol, with little or no colocalization with the plasma membrane (Fig S5C). This stands in contrast with findings in the SH-SY5Y cells, which had both plasma membrane and cytosolic enrichment of GFP-GSDME (Fig S5A & Supp video 1). The appearance of GSDME puncta in the distal and proximal neurites preceded PI uptake, indicating cell death, by several hours (Fig 2I-J). To assess whether the neurite associated puncta represent local sites of activated GSDME, we pre-treated the neurons with zVAD-FMK (caspase inhibitor), at a concentration that blocked GSDME cleavage (Fig S3A-B). Early toxin-induced puncta formation was inhibited by zVAD-FMK ( Fig   S5A, S5D-E & Supp video 1. This data suggests that in neurons, caspase dependent GSDME localization may occur earlier and to a larger extent to intracellular membranes than to the plasma membrane.
Previous work has shown that mitochondrial toxins such as rotenone, 6-OHDA and others cause cardiolipin to be externalized to the OMM in both primary cortical neurons and SH-SY5Y cells 33,34 .
Given the ability of GSDME to bind CL 11 , we next tested the hypothesis that GSDME may be localized to mitochondria along the neurites. In live cell imaging studies of cortical neurons, cells were co-transfected with GFP-GSDME and with mCherry-OMP25 to allow visualization of the outer mitochondrial membrane (OMM). We analyzed the fraction of GSDME that colocalized with OMP25 in the distal axons and found raptinal treatment resulted in a 1.5-fold increase enrichment of GSDME on axonal mitochondria and soma ( Fig. 3A-C and S6A-B). In addition to raptinal, the mitochondrial toxin rotenone also caused rapid co-localization of GFP-GSDME with mCherry-OMP25+ mitochondria (Fig S6C-E). Immunoblot analysis of fractionated lysates from raptinaltreated SH-SY5Y cells (Fig S6F) and primary cortical neurons revealed a three-fold enrichment endogenous cleaved GSDME in mitochondria compared to cytosolic fractions (Fig S6G-J). GSDME colocalization with mitochondria was also associated with release of the intermembrane/intercristae space protein, cytochrome-c, into the cytosol (Fig S6 I, K-L). To validate the cardiolipin-dependent docking of GFP-GSDME on mitochondria we knocked down cardiolipin-synthase (CLS-1) in primary neurons. CLS-1 knocked down neurons showed decreased GFP-GSDME puncta formation post toxin treatment (Fig S7 A-C). This data suggests that following activation, GSDME localizes to neuronal mitochondria in a cardiolipin-dependent manner.
We then asked whether local toxin-exposure can induce GSDME activation in axons, without involvement of the soma. We plated mouse cortical neurons in microfluidic chambers, which allowed for selective treatment of the axonal compartments with toxin (Fig 3D). We found that raptinal caused GFP-GSDME puncta formation and mitochondrial colocalization that was specific for the axonal compartment and did not occur in the untreated soma or proximal neurites of the same cell (Fig 3D-F). These data suggest that mitochondrial stress leads to enrichment of GSDME on neuronal mitochondria and can occur locally in axons.
We next used high resolution structured illumination microscopy (SIM) of neurons transfected with full-length GFP-GSDME and treated with raptinal. This analysis revealed robust puncta formation that colocalized with the outer mitochondrial membrane (OMM) marker mKate-OMP-25, but not with the matrix marker Cox8-mCherry (Fig 3G-H; Supp Videos 2-3). These results suggest that toxin treatment targets activated GSDME to mitochondrial membranes, and not the matrix.

Neurotoxins induce GSDME cleavage and mitochondrial localization in human iNeurons
Human iPSC derived neurons (iNeurons) are postmitotic, electrically active, and thus more representative of human neurons than SH-SY5Y neuroblastoma cells 35 . Here, we generated glutamatergic neurons that are most similar to layer 2/3 glutamatergic neurons of the cerebral cortex 36 . We investigated whether GSDME is similarly activated in these cells. We found raptinal treatment induces a dose dependent GSDME cleavage in iNeurons (Fig S8A). The activation of GSDME in iNeurons was blocked in the presence of the caspase inhibitor zVAD-FMK (Fig S8B).
Human iNeurons transfected with GFP-GSDME and mCherry-OMP25 also showed robust mitochondrial colocalization upon toxin treatment, with a large number in the distal neurites ( Fig   S8C-E).

Gsdme deficiency protects neurons from mitochondrial damage
We next asked whether GSDME enrichment on neuronal mitochondria potentiates damage to these organelles. To assess the functional consequences of GSDME mitochondrial colocalization in neurons, we performed TMRM staining following toxin treatment. TMRM is a voltage sensitive dye sequestered by healthy, polarized mitochondria. TMRM rapidly loses fluorescence following mitochondrial damage and membrane depolarization 37 . Treatment of wild-type primary neurons with raptinal, the complex I inhibitor rotenone, or the complex II inhibitor antimycin-A, leads to dose dependent mitochondrial depolarization and loss of TMRM stain. Depolarization was partially rescued in Gsdme KO primary cortical mouse neurons and SH-SY5Y (Fig 4A and Fig   S9A-D). Though the timing and extent of rescue differ between cortical neurons and SH-SY5Y cells, toxin-treated GSDME KO cells consistently had higher TMRM staining than WT counterparts. These experiments suggest that downstream of an initial mitochondrial insult (i.e., toxin exposure), GSDME potentiates or accelerates mitochondrial damage. To assess how GSDME impacts mitochondrial membrane integrity we transfected WT and KO cortical neurons with mKate2-OMP25 (OMM protein) and treated with toxin. After 2h and 4h of treatment (but not by 1h), Gsdme KO neurons had preserved OMP-25 signal, suggesting a sparing of the OMM relative to WT cells (Fig 4B-C). To directly visualize the structural integrity of mitochondria, we performed transmission electron microscopy (TEM) of WT and Gsdme KO neurons treated for 1h with raptinal. Analysis of TEM images revealed that raptinal-treated WT mitochondria had less pronounced cristae and increased fragmentation (reduced length) compared to those in Gsdme KO neurons (Fig 4D-E). Raptinal-treated WT neurons also displayed a high percentage of mitochondria with damaged inner or outer membranes, while Gsdme KO neurons were almost completely rescued (Fig 4F). This suggests that GSDME increases mitochondrial membrane damage downstream of toxin-mediated electron transport chain disruption.
Immunoblots for cyt-c revealed that toxin treated GSDME KO SH-SY5Y displayed a significantly higher mitochondrial to cytosolic cyt-c ratio, relative to WT cells (Fig S6 K-L). This indicates that mitochondria from toxin-treated KO cells had less OMM damage (i.e., cyt-c release) compared to WT cells. Consistent with less mitochondrial depolarization and cyt-c release, GSDME KO SH-SY5Y also exhibited reduced caspase-3 activation relative to WT cells (Fig S10A-C). Though this feed forward amplification of caspase-3 by GSDME has been described in other cell types (Fig   2A) 38 , we document it in SH-SY5Y cells exposed to complex I inhibitors and PD-associated toxins (e.g., rotenone and 6-OHDA). Gsdme KO cortical neurons treated with raptinal also had partially reduced caspase-3 activation relative to WT neurons (Fig S10D-E) albeit to a lesser extent than the SH-SY5Y cells. When taken together, these data indicate that GSDME participates in a positive feedback loop to amplify mitochondrial damage and downstream caspase-3 activation in SH-SY5Y and primary mouse neurons.
Destruction of axonal mitochondria or disruption of trafficking to distal axons is an early step in many neurodegenerative diseases 1,3 . If GSDME destroys mitochondrial membranes following toxin exposure, we reasoned that fewer mitochondrion would resume normal trafficking after toxin withdrawal. To determine the role of GSDME in impacting mitochondrial trafficking, we transfected primary neurons with mKate-OMP25 and took high timescale images in WT and Gsdme KO cells treated with toxins. At baseline in primary mouse neurons, we noticed no differences in mitochondrial movement (percent motile) between the two genotypes (Fig 4G-H). Treatment of WT cultures with the mitochondrial toxins raptinal and rotenone caused a striking arrest of motility at 1h and 2h post-treatment, as displayed in kymograph analyses (Fig 4G). This arrest was reduced in Gsdme KO cells, which had more motile mitochondria at these timepoints (Fig 4H).

GSDME regulates neurite integrity and local mitochondrial potential
Axons require a constant supply of energy to maintain electrical excitability, structural integrity and synaptic function 1 . Given that GSDME promotes mitochondrial depolarization and reduces motility following toxin exposure, we next hypothesized that GSDME KO neurons would have reduced degeneration of distal axonal processes. When treated with toxins, WT neurons displayed a dose dependent increase in the ratio of depolymerized to polymerized beta III-tubulin (Fig S10F-G). Higher levels of depolymerized tubulin indicate microtubule disassembly and axonal degeneration 39 .
Following 30 min (raptinal, antimycin-A) or 2h (rotenone) treatment with toxins, cells were incubated in fresh media for 8h and then stained for beta-III-tubulin (Tuj1) (Fig 5A). Toxin-treated WT cells exhibit a higher ratio of depolymerized (clumpy and round) to total beta-III-tubulin; GSDME knockout cells demonstrated a striking preservation of axonal processes containing polymerized tubulin (Fig 5B-D). At these timepoints, release of LDH from WT and KO neurons was minimal, indicating that GSDME increases axonal destruction prior to overt cellular necrosis ( Fig S10H, compare with Fig 2H). Incubation of toxin-treated WT cells with zVAD-FMK also reduced the microtubule depolymerization index, phenocopying the effect of GSDME knockout ( Fig S10I). Caspase inhibition rescue of neurite retraction also accords with findings from prior studies 3,40 . Collectively, these experiments suggest that the caspase-3/GSDME axis is necessary to disrupt mitochondrial health and motility and to drive neurite loss before it contributes to overt cell death at the soma (i.e. PI uptake, LDH release).
We next asked whether the N-terminal GSDME fragment (N-GSDME) is sufficient to drive neuronal dysfunction. Expression of N-GSDME in primary mouse neurons caused localization to mitochondria in cell bodies and axons in the absence of any mitochondrial toxin treatment (Fig   S11A-B). SIM microscopy analysis revealed overlap of GFP-N-GSDME and OMP-25 in both neurons and SH-SY5Y cells. However, only in SH-SY5Y cells did we observe GFP-GSDME enrichment on plasma membrane (Fig S11B-D). Expression of N-GSDME in primary cortical neurons was sufficient to cause mitochondrial potential loss (Fig S11E-F) and neurite damage as measured by Tuj1 staining (Fig S9G-H). Thus, N-GSDME expression phenocopies results obtained following chemical activation of GSDME using toxin treatment.
Using microfluidic chambers, we found that GSDME can be locally activated in axons and rapidly colocalizes with mitochondria (Fig 3D). To determine the effect of this local GSDME activation on axonal mitochondrial health, we again plated neurons in microfluidic chambers and stained with TMRM (Fig 5E). Following raptinal treatment of the axonal chamber, we observed dosedependent TMRM decreases only in the treated axons (Fig S10J-K). Next, we examined WT and GSDME KO axonal compartments treated with toxins for differences in TMRM staining. Raptinal caused a rapid and pronounced loss of TMRM positivity, which was partially rescued in GSDME KO neurons (Fig 5E-F). While long term raptinal exposure leads to total TMRM loss, brief (30min-2h) treatment causes mitochondrial depolarization that is GSDME-dependent (Fig 5G). These data demonstrate that GSDME can be activated locally to destroy mitochondria, in the absence of overt changes at the cell body.

ALS/FTD-associated proteins induce GSDME activation in neurons
Neurodegenerative conditions are characterized by early axonopathy that precedes loss of neuronal soma 26,27 . Given that GSDME can drive axonal mitochondrial damage and disrupt structural integrity, we examined whether neurodegeneration-associated proteins would similarly activate GSDME. ALS and FTD are devastating neurodegenerative conditions characterized by progressive and ultimately fatal neuronal cell death. Both conditions are characterized by early mitochondrial damage in neurons and axon loss 26,41,42 . We hypothesized that GSDME could be activated in these settings and potentially contribute to neuronal dysfunction.
To test whether ALS/FTD associated proteins cleaved GSDME in our in vitro system, we focused on the role of TDP-43 and the C9ORF72 hexanucleotide expansion-associated dipeptide repeat protein (DPR) PR-50. TDP-43 is an RNA binding protein that forms neuronal inclusions in both familial and sporadic ALS/FTD brain and spinal cords 43 . Prior work has shown that TDP-43 aggregates bind mitochondria, leading to complex I inhibition and mitochondrial depolarization [44][45][46] . The DPRs produced from mutated C9ORF72 have also been shown to impair mitochondrial function (complex V activity), leading to caspase-3 dependent axon loss 39,47,48 . Given that these neurodegenerative proteins are associated with direct mitochondrial damage and caspase-3 activation, we hypothesized that GSDME may be involved in downstream neuronal dysfunction mediated by these proteins.
Mouse cortical neurons were transfected with TDP-43, PR-50 or RFP control plasmids to model neurodegeneration in a dish 39 .Co-expression of TDP-43 or PR-50 with GFP-GSDME, robustly increased GFP-GSDME puncta (Fig 6A-B). These puncta were enriched on neurite mitochondria, mirroring results obtained using complex I inhibitors. Treatment of WT cortical neurons with lentiviruses encoding control (GFP), TDP-43 or PR-50 under control of a Synapsin-I promoter, led to cleavage of endogenous GSDME at 72h post-transduction, indicating its activation (Fig 6C   and Fig S12A).

TDP43 and PR-50 induced mitochondrial damage and neurite loss are GSDME-dependent
We next asked whether GSDME could contribute to neuronal dysfunction caused by ALS/FTD associated proteins in mouse neurons. We found that WT neurons transduced with TDP-43 and PR-50 exhibited both significant neurite loss and mitochondrial depolarization indicated by loss of TMRM staining at 72h and 96h post-treatment ( Fig. 6D-E and Fig S12B). GSDME KO cells had reduced mitochondrial depolarization compared to WT neurons (Fig 6D-E and Fig S12B). These experiments indicate that ALS/FTD associated proteins can cause GSDME activation, mitochondrial localization and mitochondrial depolarization that partially relies on GSDME.
Human iNeurons are often used to model disease in vitro 35 , and express full-length GSMDE at baseline (Fig S8A). To ask whether neurodegeneration-associated proteins could activate GSDME in human cells, we co-transfected cortical iNeurons with plasmids encoding WT TDP-43 (or iRFP control), GFP-GSDME and mKate-OMP25. TDP-43 transfected neurons exhibited robust GFP-GSDME puncta that colocalized with mitochondria (Fig S12C-D). As in mouse neurons, we found that lentiviral transduction with FLAG-tagged TDP-43 (at MOI 4 and 8) led to cleavage of endogenous GSDME relative to GFP transduced controls (Fig S12E). When taken together, these data suggest that a diverse range of neurotoxic stimuli-mitochondrial toxins, PR-50 and TDP-43 can lead to downstream GSDME activation and mitochondrial colocalization in both mouse and human neurons.
Given that GSDME KO cortical neurons had partially rescued mitochondrial depolarization, we assessed whether these neurons would also have spared neuritic processes. We took two complimentary approaches to answer this question. First, we transfected neurons with GFP and either TDP-43 or PR-50 plasmids. We then measured neurite area of transfected neurons using the GFP expression as a marker. Compared to WT neurons, GSDME KO cells transfected with PR-50 or TDP-43 had preserved neurite area (Fig S12F-I). Next, we used lentiviruses encoding either PR-50 or TDP-43 and measured resulting microtubule depolymerization index in cortical neurons at 72h (TDP-43) or 96h (PR-50) post-transduction. GSDME KO neurons were protected from neurite loss, with results mirroring those obtained using mitochondrial toxins (Fig 6F-I). At these timepoints there was minimal LDH release from WT and KO cells compared to untransduced controls (Fig 6J-K). This indicates that TDP-43 and PR-50 mediate GSDMEdependent axonal loss occurs prior to frank cellular necrosis. Collectively, these experiments suggest that ALS/FTD proteins engage GSDME to drive neuritic mitochondrial dysfunction and axon loss.

GSDME impacts disease progression in the SOD1 G93A ALS mouse model.
We next tested the hypothesis that GSDME could drive neuronal dysfunction in an animal model of neurodegeneration. ALS has been linked to mutations in the human superoxide dismutase 1 (SOD1) gene. Human SOD1 G93A transgenic mice develop progressive motor neuron loss in the ventral spinal cord and paralysis 49 . Mutant SOD1 proteins have been shown to induce mitochondrial dysfunction, impaired ATP production, inefficient calcium buffering and increased apoptosis of motor neurons 28,29,50 . To assess the relevance of GSDME in vivo, we first probed spinal cord lysates from the SOD1 G93A mouse model of ALS for GSDME activation. We detected GSDME cleavage products in spinal cords of symptomatic SOD1 G93A mice (P140), which was further increased by late-stage (P160) (Fig 7A-B, Fig S13A-B). Cleaved GSDME was not detected pre-symptomatic P82 animals (Fig S13A). Given this activation pattern, we hypothesized that GSDME may contribute to disease progression in the SOD1 G93A model. We created mice heterozygous for the SOD1 G93A transgene and either wild-type (SOD1 G93A GSDME WT) or knockout for GSDME (SOD1 G93A GSDME KO). We confirmed that experimental animals had similar copy numbers of the SOD1 G93A transgene irrespective of GSDME status by RT-qPCR; non-transgenic animals did not express SOD1 G93A (Fig S13C).
We found that knockout of GSDME significantly extended survival and delayed disease progression in mice containing the SOD1 G93A transgene (Fig 7C). Mean survival for SOD1 G93A GSDME WT mice was 159+1.9 days, while that of SOD1 G93A GSDME KO counterparts was 171+2.5 days (Fig 7C). Female SOD1 G93A GSDME KO animals received a slightly greater survival benefit relative to female SOD1 G93A GSDME WT mice compared to males (Fig 7D-E & Fig S13F).
As a measure of disease on set, the age to maximum body weight were unaffected by GSDME genotype in SOD1 G93A animals (Fig 7F & Fig S13D). However, disease progression-as measured as time from maximum body weight until the time point of euthanasia-was significantly extended in SOD1 G93A GSDME KO animals relative to SOD1 G93A GSDME WT littermates ( Fig   7G). Loss of grip strength was also significantly delayed in SOD1 G93A GSDME KO compared to SOD1 G93A GSDME WT mice (Fig 7H). No baseline differences in grip strength were recorded for non-SOD1 transgenic GSDME WT and GSDME KO animals (Fig S13E). These results indicate that GSDME deficiency impacted the symptomatic progression phase in this animal model, leading to an overall motor and survival benefit (supplementary video 4).
Consistent with the rescue in grip strength and survival, motor neurons in the ventral horn were preserved in SOD1 G93A GSDME KO mice at P150 (late-stage) relative to age-matched SOD1 G93A GSDME WT littermates (Fig 7I-J). We also looked at makers for glial activation in the spinal cord, as microgliosis and astrogloisis are hallmarks of disease progression in the SOD1 mouse model of ALS and in human ALS patients [51][52][53][54][55] . We observed that GSDME KO mice showed decreased intensity of GFAP+ astrocytes (astrogliosis) in the ventral horns of SOD1 G93A animals (Fig S14A-B). SOD1 G93A GSDME KO animals also showed decreased microgliosis, as measured by the number of Iba1+ positive cells in the ventral horn (Fig S14C-D) and levels of the microglial lysosomal activation marker, CD68, compared to those in SOD1 G93A GSDME WT mice (Fig S14E-F). When taken together, this data demonstrates that GSDME is activated in a classic mouse model of ALS and drives pathology-namely neuronal loss and gliosis-that contributes to decreased motor function and survival.

GSDME mediates neurite loss in ALS patient IPSC-derived motor neurons
To determine potential relevance to human disease, we mined genome-wide expression profiling data of laser capture microdissection-enriched motor neurons (MNs) from patients with sporadic ALS (sALS) 56 . We found that GSDME was significantly upregulated in neurons from sALS patients relative to age-matched healthy controls (Fig S15A). This data indicates that GSDME protein is present in the human CNS and may be relevant in the setting of neurologic disease.
We next investigated whether GSDME may contribute to neuronal dysfunction in human ALS patient iPSC-derived motor neurons. To model human neurodegeneration in vitro, we cultured a control iPSC-derived MN line (1016A) and an ALS iPSC-derived MN line (TDP43 G298S ). Challenge of these iMNs with a proteasome inhibitor (MG132) or ER-stress toxins (thapsigargin and tunicamycin) led to dose dependent neurite loss and cell death; these effects are exacerbated in the mutant TDP43 G298S line (Fig S15-B 57 . We next determined if knockdown of GSDME expression in human iPSC-derived motor neurons would rescue this ER-stress driven neurite loss We next found that TDP43 G298S motor neurons transduced with lentivirus encoding scrambled (control) shRNA were highly vulnerable to neurite destruction caused by MG132, tunicamycin or thapsiagargin (Fig 8A, Fig. S15B-E). By contrast, knockdown of GSDME using lentiviral delivery of two targeting shRNAs (sequence #1 and #3) led to significant sparing of neurite loss in TDP43 G298S iMNs (Fig 8, S15 F-G). Rescue due to GSDME knockdown was also observed in control 1016A (WT) iMNs treated with ER toxins or MG132-though to a smaller extent than in susceptible TDP43 G298S iMNs (Fig S15 A-E). Collectively, these results support that GSDME drives pathology downstream of ER-stress and proteasome inhibition in WT MNs, and to a much greater degree, in the vulnerable TDP43 G298S genetic background. Thus, GSDME deficiency can rescue neurite loss in a patient-derived ALS model.

Discussion
Mitochondrial dysfunction is an important and early hallmark of neuronal injury. Here we describe GSDME as a critical regulator of mitochondrial damage, neurite loss, and death in neurons downstream of multiple classes of neurotoxic stimuli. Both mouse and human neurons express GSDME, and this protein is activated following exposure to mitochondrial toxins and ER stressors.
Several such toxins (e.g. rotenone and MPTP) are linked to neurodegeneration in humans, and others can be used to model features of disease in vitro and in mouse models 58,59 . GSDME is also activated following neuronal expression of ALS/FTD associated proteins, TDP-43 and PR-50. Activated GSDME rapidly colocalizes with axonal mitochondria prior to overt cell death, driving both mitochondrial damage and neurite loss. We further show that GSDME plays a significant role in the SOD1 G93A mouse model of ALS and in an iPSC derived motor neuron model of neurodegeneration, thus identifying this axis as a therapeutic target.
We uncover several aspects of GSDME biology unique to neurons that differ from previous studies in cell lines or primary immune cells. We show that GSDME mediates axonal mitochondrial damage and local neurite loss. In neurons, active GSDME primarily localizes to mitochondrial membranes and mediates mitochondrial destruction and neurite loss, in the absence of significant plasma membrane localization and rupture (as assessed by LDH release).
There may be multiple reasons behind the resistance of neurons to membrane rupture. For example, the neuronal plasma membrane lipid composition may be uniquely resistant to N-GSDME insertion, or may harbor components that limit its activity in neuronal cell membranes (akin to the relationship between XIAP and CASP3). It is also possible that neuronal plasma membrane damage from GSDME activation is rapidly repaired, while delicate axonal processes are not spared. Such a phenomenon -ESCRT complex repair of gasdermin-mediated plasma membrane damage -has been described following GSDMD activation in myeloid cells 60,61 . In macrophages, this is important for cytokine secretion as it allows "hyperactivated" living macrophages to engage GSDMD mediated IL-1 release without overt cell death 60,61 . In the CNS, there may be a physiologic benefit in destroying distal processes while keeping the soma intact.
For example, GSDME mediated neurite pruning may be crucial to either development or host defense (e.g. to stop the spread of viral or microbial pathogens). However, in the setting of progressive neurodegenerative pathology (such as in ALS), GSDME may be inappropriately activated to drive disease progression and contribute to neuroinflammation. Our findings thus expand the biological role for GSDMS in the CNS. Elucidating the physiological contexts (e.g. development or infection) of GSDME mediated neurite loss would be an important future direction.
Prior work explored GSDME activation in cancer cell lines, focusing primarily on its contribution to overt cellular necrosis 11,25,38 . Activated caspase-3 cleaves and activates GSDME, converting noninflammatory apoptotic cell death to inflammatory pyroptosis. GSDME colocalizes with mitochondria in HeLa cells treated with DNA-damaging agents (e.g. UV radiation, etoposide) and mediates cytochrome-c release to further amplify caspase-3 processing and necrosis 10,38 .
Oxygen or glucose deprivation also leads to caspase-1/Bid/caspase-3 activation in cortical neurons; it has been suggested that GSDME may contribute to cytochrome-c release and cell death in this context 62 . More recent work in a mouse model of macular degeneration and a murine retinoblastoma cell line has implicated GSDME in amplifying cytochrome-c release and capsase-3 activation in sensory neuroepithelium 23 . We establish this positive feedback loop in primary neurons and map its neuron-specific cell biology-namely localized GSDME activation in axons, mitochondrial damage, disruption of mitochondrial trafficking and neurite-loss.
Spatially restricted GSDME activity in axons (Figs 3 & 5) raises the possibility that GSDME is a key downstream effector of caspase-3 mediated axonopathy and neuronal demise. Caspase-3 inhibition has been shown to protect neurons from age or disease related axon loss and cell death 3,63-65 . In neurodegeneration, a unique combination of environmental exposures and gene expression differences may predispose certain neurons to activate the caspase-3/GSDME axis. Neurons may utilize multiple pore-forming molecules to regulate mitochondrial membrane integrity; namely upstream pro-apoptotic BCL-2 family members (e.g. Bax, Bak, Bid), the mitochondrial permeability transition pore (MPTP) and in concert with GSDME. Caspase-3 cleaves GSDME, allowing for N-terminal oligomerization and insertion into lipid membranes.
Following mitochondrial colocalization, GSDME subsequently drives mitochondrial depolarization, cyt-c release, and enhanced caspase-3 activation. Thus, GSDME is a versatile lipid-targeting pore forming molecule that may have overlapping functions with Bax/Bak families as well as MLKL-the pore-forming executioner of necroptosis 66,67 . How these distinct families of pore-forming molecules operate in a cell-type specific manner in neurodegeneration, or work in concert to form compensatory signaling loops is poorly understood, and an important area of future investigation. Given the link between mitochondrial toxins and stress to quality control programs (mitophagy and mitochondria derived vesicles), it would also be important to see how these mechanisms function either in series or in parallel to GSDME activation.
We found that expression of the active N-terminal GSDME fragment in neurons is sufficient to cause mitochondrial dysfunction and neuritic retraction independent of any other stimuli, indicating that it may have a preference for mitochondrial membranes in neurons. Indeed, biophysical assays have shown that N-terminal GSDME (as well as GSDMD) binds cardiolipin with much higher affinity than it does phosphatidylcholine, phosphatidylinositol or other lipids on the OMM 11,13 . Mitochondrial stress can externalize cardiolipin from the inner to outer mitochondrial membrane 68,69 . Our work demonstrates that in neurons, GSDME rapidly localizes to mitochondria prior to axon degeneration and neuronal cell death. Knockdown of cardiolipin synthase (CLS1) reduces GSDME colocalization with mitochondria, suggesting a role for cardiolipin in homing of GSDME to mitochondria. However, this mainly occurred at early time points (Fig S7)-it is unclear whether this partial rescue is due to incomplete knockdown or the potential ability of GSDME to bind other ligands present in the OMM. Investigating which other lipids or proteins regulate GSDME binding to the OMM will be an important avenue of future mechanistic investigation.
Destruction of axons is an early step in conditions such as neurodegeneration, neuropathic pain, and stroke 26,27,70 . Our findings using imaging and microfluidic compartments demonstrate that GSDME activation can occur locally in axons and drive local mitochondrial damage, without involving the cell body. Thus, our study positions GSDME as a molecule that may work alongside calpains, SARM1, KIF2A, MAPK-JNK, loss of STMN2 and other effectors to disrupt axons.
Though phosphorylation and palmitoylation of GSDME residues can modulate its activity, there are no known GSDME regulators apart from upstream proteases (caspase-3 and granzyme-B) 25,38,71 . Calpains are proteases with cleavage activity linked to axon degeneration 72 . SARM1 has been shown to regulate the extent of GSDMD-driven pyroptosis in immune cells 73 . The MAPK-JNK signaling axis promotes SARM1 dependent axonal degeneration in vivo, but is also an upstream regulator of GSDME activity in cancer cell lines [74][75][76] . It will be important to determine if SARM1, calpains, JNK or other mediators of axon degeneration regulate GSDME function in a common pathway to destroy neuronal processes.
We further showed that GSDME is activated by neurodegeneration associated proteins (TDP-43 and PR-50). That both mitochondrial toxins and aggregated proteins evoke GSDME signaling is perhaps not unexpected. TDP-43 has been shown to bind and damage mitochondria, leading to release of DNA and activation of STING signaling to drive cell death 45 . C9ORF72 DPRs directly inhibit mitochondrial complex V, leading to membrane depolarization and neuronal death 77 . Motor neurons of patients and mice carrying SOD1 mutations, have impaired electron transport chain function, disrupted mitochondrial ATP production, inefficient calcium buffering, and enhanced caspase-3 activation 28,50,55 . That mutant SOD1 aggregates colocalize with mitochondria further suggests a directly toxic effect 29 .
How neuronal GSDME gene expression is regulated remains to be determined. Transcriptional data from micro dissected motor neurons showed an upregulation of GSDME in patients with ALS compared to age-matched controls. Prior work has shown that tumor suppressor gene p53 can bind to intron 1 of the GSDME gene to induce its expression, and p53 knockout mice display reduced levels of GSDME in the GI epithelium 19 . A recent study showed that p53 drives C9ORF72-dependent cortical neuron death in a mouse model of FTD/ALS 39 . As suggested for other tumor suppressor genes (e.g. ATM, p53, Bax and WWOX), GSDME may in one context suppress cancer growth and in another, inappropriately promote neuronal injury and inflammation [78][79][80] .
When we knocked out GSDME in the SOD1 G93A transgenic background, we observed a slowing of disease progression. This data functionally links the activation of GSDME with neurodegeneration in a mouse model of ALS. We found GSDME was activated (cleaved) in spinal cords at late stages of disease and its deficiency led to decreased microglia and astrocyte activation. Previous studies have found that activation of GSDME impacts mouse models of tumor progression, chemotherapy induced lung damage, cisplatin-induced kidney injury, macular degeneration and inflammatory bowel disease 22,23,25,81,82 . In these settings, knockout of GSDME not only reduces cell death, but also reduces local inflammation. Mapping how exactly GSDME promotes in vivo neuroinflammation-either via neuronal release of pro-inflammatory cytokines, damage-associated molecules (e.g. HMGB1 and ATP), or other secreted factors to recruit glia or CNS-infiltrating immune cells-will be an important step in understanding its role in disease pathophysiology.
We extended our in vivo results by employing an in vitro human model of ALS-assessing neurite loss in ALS iPSC-derived motor neurons that carry a TDP-43 mutation (Fig 8). In these cells, neurite loss was rescued by GSDME knockdown, supporting that this pore-forming molecule may contribute to ALS pathology. These iPSC-derived in vitro systems are important tools to further understand the mechanisms by which GSDME is activated in human disease and affects neurodegeneration.
In conclusion, our work positions GSDME as an intrinsic executioner of axon degeneration and cell death in neurons. When activated by diverse neurotoxic stimuli, GSDME drives features of neurodegeneration in vitro-destruction of mitochondria, enhancement of caspase-3 activation and neuritic retraction-and contributes to disease progression in two ALS models. Recently, small-molecule inhibitors of GSDMD activation have been identified 83,84 , though it is unclear whether GSDME is similarly druggable. Future work should determine the feasibility of targeting GSDME expression or function. Ultimately, determining how multiple cell death axesgasdermins, caspases, calpains, and necroptotic machinery-contribute to neuronal cell death may suggest new therapeutic strategies for neurodegenerative diseases.

Limitations of this study
In vitro analysis using mouse and iPSC-neurons allows for well controlled pharmacological and genetic manipulation, careful microscopic examination and discovery of exciting new aspects of neuronal cell biology that are not possible to observe currently in vivo. However, cultured mouse and human iPSC-derived neurons do not recapitulate the complexity of the nervous system. In vivo, it is possible that there are intrinsic compensatory feedback loops and non-cell autonomous mechanisms influence GSDME signaling. Such interactions may not be possible to model in pure neuronal cultures. Further, biochemical purification of mitochondria in cultured systems may be contaminated by plasma membrane fractions. Additional experiments controlling for this possibility, such as neuronal plasma membrane fractionation, would strengthen the work. We demonstrate that GSDME is engaged in neurons after toxin or FTD-associated protein exposure, in several cellular contexts lending strength to our conclusions. Future studies employing mouse models of ALS/FTD coupled with GSDME knockout will be necessary to further elucidate the role of GSDME in a more physiologic context of neurodegeneration. Lastly, experiments showing in vivo human GSDME cleavage and immunostaining are inherently limited by time resolution-cell death proteins are difficult to detect in vivo due to their transitory nature. Once a cell dies, its mRNA and protein may not be readily detectable by standard methodologies. Increased sampling of tissues from mouse models and human patients will elucidate when and how this molecule is regulated during disease.

SOD1 G93A Breeding and Behavioral Assessments
Breeding: SOD1 G93A mice (JAX #002299) were originally acquired from Jackson Laboratory (Bar Harbor, ME). The SOD1 G93A GSDME KO mice were obtained by crossing male mice carrying the SOD1 G93A transgene with female GSDME KO (JAX #032411) mice for 3 generations. The control SOD1 G93A GSDME WT mice, were obtained by crossing SOD1 G93A mice with wild type female mice having a B6NJ background (JAX #005304), also for 3 generations. This breeding strategy controlled for any strain background related phenotypic differences.
All offspring were tested for equivalent SOD1 gene copy numbers by qPCR (forward primer: CAGTAACTGAGAGTTTACCCTTTGGT; and reverse primer: CACACTAATGCTCTGGGAAGAAAGA. Mice that had SOD1 signal drop-off of more than 30% from control SOD1 G93A mice were considered as low copy number animals and were excluded from the study. Male and female mice were used for all experiments and experimental groups were sex balanced. Animals were provided with food and water ad libitum.
Weight Measurement: Weights of all mice carrying the SOD1 G93A transgene were measured biweekly from week 7 to week 21. Disease onset was defined as the day of peak weight while disease progression was defined as the number of days between disease onset to euthanisia. The top three pulls per time point were averaged and used for data analysis. 12 or more mice per genotype, per sex, were used for weights and grip strength analysis.
Survival: Each mouse was monitored daily after symptom onset. Mice that were unable to rear were given hydrogels and wetted food pellets at the bottom of the cage. Euthanasia time points for each mouse were determined by the inability to successfully right itself when flipped on either of its sides within 30s.

Human iPSC-derived cortical neuron culture and plating
NGN2 tetracycline-inducible iPSC neuron lines were a gift from the Tracey Young-Pearse lab and were generated using a lentiviral transduction protocol as previously described 86 . Plates were coated with poly-orinatine and laminin (10 ug/mL poly-ornithine, 5 ug/mL laminin) 24

Drugs and treatments
Raptinal (cat# SML1745), rotenone (cat# R8875), antimycin-A (cat# A8674) and 3-Nitropropionic acid (cat# N5636) were purchased from Sigma and resuspended in DMSO to make stock solutions. For raptinal treatments, cells were exposed to final concentrations ranging from 1-10uM for 1h, and then incubated with fresh media and assessed at later timepoints. For antimycin-A treatments, cells were exposed to final concentrations ranging from 5-20 uM for 30 min and then incubated with fresh media. For rotenone and 3-NP treatments, cells were exposed to working concentrations ranging from 10-30uM (Rotenone) and 1-3 mM times with warmed plain NBM, and DNA/lipofectamine mix (100uL/well for a 24 well plate) was added to each well and incubated for 1h at 37C. Following incubation, each transfected well was washed 3 times with fresh plain NBM and the conditioned media was added back to each well.
Neurons were then incubated for 2-3 days at 37C prior to imaging and analysis.

Plasmids
All plasmids used in this study for transfections are as follows:

Lentivirus transductions of primary neurons
Neurons were transduced overnight with lentiviruses encoding control (eGFP) or PR-50 and TDP-43 proteins as described previously. 39 Briefly, primary neurons plated in 96-well plates at a density of 50-75,000 cells/well were grown for 72 hours. The neurons were then treated with tittered viruses at MOIs of 2 or 3. Transduction volume was 40uL/well. Following overnight incubation with virus, neurons were washed 3x with warmed complete NBM and followed for TMRM staining, immunoblot analysis or Tuj1+ staining as described below.

Live cell imaging of neurons
For live-cell imaging of cortical neurons, neuronal cultures grown on glass bottom dishes at a density of 175,000 cells/cm 2 were imaged on a DMI8 Zeiss microscope. The microscope was equipped with an environmental chamber that was supplied with humidified 5% CO2 and maintained at 37C. Images were acquired with an Andor Zyla sCMOS camera via a 20× Plan Apo objective. To monitor GSDME enrichment on mitochondria, images from neurons co-transfected GFP-GSDME and mKate2-OMP25 (referred to as mito-RFP) were captured every 15 minutes following treatment with toxins (raptinal and rotenone). To monitor mitochondrial motility, each well of neurons (350,000 cells/well) were sparsely transfected with mKate2-OMP25 (referred to as mito-RFP) and cytosolic GFP. Two days following transfection, the neurons were treated and imaged. Images of whole neurons (all visible neurites) were captured every 6s. Kymographs were generated from 3-to 5-min time-lapse movies and analyzed with a custom-written ImageJ macro for percent motility mitochondrial density, and length. High resolution 3-5 min timelapse movies were taken at 30 min, 1h and 2h post-raptinal treatment.

Cell death assessment (LDH and PI assays)
Neurons were plated at 7.5 x10 4 cells/well of a tissue culture-treated black 96 well plate coated with laminin and poly-L-lysine with optically clear flat wells 7d prior to experimentation. Propidium iodide or SYTOX green were diluted in complete NBM to a final concentration of 1.5uM or 1uM respectively, and added to neurons during treatment with toxin incubation as described above.
Positive control wells were lysed by the addition of 5 ul of 10x lysis buffer (10% v/v Triton X-100) 45 min prior to the desired endpoint for analysis. Propidium iodide uptake was analyzed using the IncuCyte S3 Live cell analysis imaging system and associated software (Sartorius V2018B

TMRM assays
Image-iT TMRM (Thermo Scientific cat# I34361) reagent was used at a concentration of 100 nM (1:1000 dilution in NBM). Primary neurons were incubated with TMRM solution for 1h at 37C, and then washed with fresh NBM. Cells were treated with toxins or lentiviruses as described above and imaged every 30 min using the IncuCyte S3 Live Cell Imaging system. Image acquisition and data analysis were performed using the IncuCyte S3 Software (Sartorius V2018B) TMRM positive neurons were scored as cells with a threshold signal greater than 2.5 red calibrated units (RCU) above background, using a Top-hat background subtraction method. Total TMRM intensity was calculated for each well and normalized to DMSO control or vehicle conditions.

RNA isolation and qPCR
For analysis of mouse brain tissue, animals were anesthetized with Avertin solution (500 mg/kg,

Mouse brain and spinal cord immunohistochemistry
Adult mice were perfused with 20mL of cold PBS and 20mL of 4% PFA. Brains or spinal cords were dissected, fixed overnight in 4% PFA at 4C, paraffin embedded and processed for IHC as described 90  Human tissues were processed and stained as previously described 91 . Briefly, slides were deparaffinized in xylene, and dehydrated in successive EtOH washes. After a brief wash in dH20, slides were then incubated for 30 min in a 70% MeOH and 30% H2O2 solution, and then washed in tap water. Microwave antigen retrieval was performed for 20 min in a citrate buffer (Vector Labs #H-3300). Slides were then cooled to room temperature, rinsed in TBS (0.1 M Tris Buffer) and subject to blocking solution (TBS/2%FBS/3%BSA) for 5 min at room temperature. Following blocking, anti-Human GSDME antibody (abcam 230482, rabbit-anti-human) was diluted 1:100 in blocking solution. Sections were incubated in GSDME primary antibody overnight at 4C in a humidified chamber. The next day, slides were washed with TBS, blocked for an additional 5 minutes and exposed to vector biotinylated anti-Rb IgG secondary antibody (Vector #BA-1000) at a dilution of 1:1000 for 1h at room temperature. Slides were then washed in TBS and Vector ABC solution (Vector # PK-4001) was applied to each slide for 1h at RT. Slides were then exposed to Vector IMPAACT DAB solution at room temperature. Following development, samples were washed in dH20, counterstained using hematoxylin (Cell signaling #14166), rinsed in tap water, dehydrated in successive EtOH and xylene rinses and mounted for imaging.

Transmission Electron microcopy
Primary mouse neurons were cultured for 7d (as described above). WT and GSDME KO cells were then treated with 5uM raptinal for 1h, prior to processing for electron microscopy. Cells were fixed for at least 2 hours at RT in a 2.5% Glutaraldehyde 1.25% Paraformaldehyde and 0.03%

Data mining of postmortem sALS motor neurons
FASTQ files were obtained through the NCBI GEO database (GSE76220) 56  PFA.

Data analysis and code availability
Enrichment analysis of GSDME on mitochondria, quantification of GSDME puncta and analysis of microtubule depolymerization was done using custom Fiji (ImageJ) macros (available at: https://github.com/IsaacChiuLab/FIJIanalysisCodes_GSDME).
Colocalization analysis of GSDME and mitochondria along neurites: For analyzing the enrichment of GSDME on mitochondria along neurites, the neurites were tracked manually. The mitochondrial intensity and the GSDME intensity were then measured along the neurites. The mitochondrial intensity was then converted to 1-D masks (by thresholding according to local contrast) while the GSDME intensity was normalized to the average intensity along the neurite. The enrichment of GSDME was then calculated as the ratio of mean GSDME signal within the mitochondrial mask to the mean GSDME signal outside the mitochondrial masks.
Colocalization analysis of GSDME and mitochondria in cell bodies and SHSY5Y cells: To analyze the enrichment of GSDME on mitochondria in cell bodies and SHSY5Y cells, the cell outline was drawn manually. The mitochondrial intensity within the cell outline was then used to create masks (by thresholding according to local contrast) and the GSDME intensity was normalized to the average intensity of the whole cell. As done in the case of neurites, enrichment of GSDME on mitochondria was reported as the ratio of mean GSDME signal within the mitochondrial mask to the mean GSDME signal outside the mitochondrial masks Quantification of GSDME puncta: To quantify the density of puncta along neurites, the neurites were manually traced. The intensity of GSDME was then measured along the neurites and normalized to the average intensity along the whole neurite. To detect GSDME puncta, the GSDME signal was then converted to a mask using a local thresholding window of 20px. The density of GSDME puncta in the cell bodies and SHSY5Y cells was quantified using a similar approach. In this case, the cells were traced manually, following which the GSDME puncta were detected by local thresholding.
Analysis of Beta-III-Tubulin (Tuj1) immunocytochemistry: The microtubule (MT) depolymerization index was measured using a custom macro adapted from 39    showing cell types (left most plot) and gasdermin genes were generated using the Allen Brain Atlas Transcriptomics Explorer tool (Allen Institute for Brain Science). (B) Representative IHC images of coronal mouse brain sections stained using anti-GSDME antibody. Scale bar: 1mm (C) Immunoblot analysis of GSDME expression in several mouse brain regions from adult wildtype and GSDME knockout mice.
(E) Representative IHC images of mouse brain sections from cortex and striatum co-stained with anti-GSDME and either anti-beta-III-tubulin (Tuj1) or anti-Iba1 antibodies. Scale bar: 20um Colocalization analysis of GSDME positive cells with markers for neuronal (Tuj1) or glial (Iba1) cells. Results are displayed as the percentage of GSDME + cells that also express either Tuj1 or Iba1. Each dot represents a coronal mouse brain section. Two coronal sections were quantified per mouse, and n= 3-5 mice were used for each co-staining experiment.
(F) Representative IHC images of temporal lobe from two healthy control patients stained with human anti-GSDME. Secondary only controls are displayed in smaller panels underneath the stained sections. Scale bar: 50um   For datasets A-C, student's t-tests were performed for comparisons between genotypes, and pvalues were adjusted using the Tukey method. Each dataset represents an average of two independent experiments + SEM. For datasets E and H two-way ANOVA was performed, followed by multiple comparisons for each group. Adjusted p-values are mentioned. For datasets B-D,E, two-way ANOVA (row factor = toxin dose, column factor = genotype) was performed, followed by multiple comparisons for each group (adj p-values, Tukey method). For datasets D-E, two-way ANOVA (row factor = time, column factor = genotype) was performed, followed by multiple comparisons for each timepoint comparing WT vs GSDME KO groups (adj p-values, Tukey method). For datasets H-I, two-way ANOVA (row factor = toxin dose, column factor = genotype) was performed, followed by multiple comparisons for each group. p-values are indicated above brackets comparing respective datasets. (C-E) Transgenic (C) mixed gender mice that were either SOD1 G93A GSDME WT (N=31 total) or SOD1 G93A GSDME KO (N=42 total) were followed for survival. These Kaplan-Meier curves are displayed separately for (D) male SOD1 G93A GSDME WT (N=17) and SOD1 G93A GSDME KO (N=22) and (E) female SOD1 G93A GSDME WT (N=14) and SOD1 G93A GSDME KO (N=20) animals.
Mice were monitored daily following onset of motor symptoms ~d140. For datasets C-F, Kaplan-Meier curves were constructed, and survival analysis was performed.
Groups were compared for statistical differences using the log-rank test (Mantel-Cox). For datasets G and J, student's t-tests were performed for comparisons between genotypes, and pvalues were adjusted using the Tukey method. For dataset H, a two-way ANOVA (row factor = time, column factor = genotype) was performed (p = 0.01), followed by multiple comparisons for each timepoint (p-values adjusted by the Tukey method). p <0.001 = *** , p <0.01 = **, p <0.05 = ***  (A) Gsdme RT-qPCR of several mouse brain regions from WT (n=3) and Gsdme knockout (n=2) mice (B) Representative IHC images of cortical mouse brain sections from WT and GSDME knockout animals stained with anti-GSDME and counterstained with DAPI.
(C) FACS-isolated brain myeloid cell transcriptomic data from the publicly available Tabula Muris database was mined for Gsdme and Gsdmd expression. TSNE plots showing cell types (left most plot) and expression of gasdermin genes (right) were generated using the CZI biohub tool (Available from: https://tabula-muris.ds.czbiohub.org/) (D) Publicly available human cortical single-cell RNA-seq (10X Genomics) data was mined for gasdermin family expression levels. TSNE plots showing cell types (left most plot) and gasdermin genes were generated using the Allen Brain Atlas Transcriptomics Explorer tool (Allen Institute for Brain Science. Available from: human.brain-map.org) (E)SH-SY5Y cells were incubated in propidium iodide (PI) containing media and treated with 5uM raptinal, 20uM rotenone or 30uM of 6-OHDA. Images were taken every 3h and quantified for PI uptake.      For all datasets two-way ANOVA (row factor = toxin , column factor = genotype) was performed, followed by multiple comparisons for each group (p-values adjusted by the Tukey method are mentioned over respective comparisons). Data represents an average of at least 2 independent experiments.   For datasets A, G, I two-way ANOVA (row factor = lentivirus treatment, column factor = genotype) was performed, followed by multiple comparisons for each group (adj p-values, Tukey method).  (A-B) GSDME and GAPDH immunoblots of spinal cord lysates from (A) pre-symptomatic stage (P82) and end-stage (P160) SOD1 G93A mice. (B) The activation ratio of GSDME is quantified as the levels of GSDME N-terminal compared to total GSDME (full length + cleaved fractions).
(C) Expression of the SOD1 G93A transgene, as assessed by RT-qPCR, was quantified for each group and genotype.This was done to confirm that the copies of this ALS-causing transgene were comparable between experimental groups.
(D) Weights of transgenic SOD1 G93A GSDME WT or SOD1 G93A GSDME KO mice were tracked over time starting at week 7 until week 21.  (E-F) Representative images of lumbar spinal cord sections from SOD1 G93A GSDME WT and SOD1 G93A GSDME KO animals at P150, stained with anti-CD68 (a lysosomal activation marker),