Endoplasmic reticulum unfolded protein response transcriptional targets of XBP-1s mediate rescue from tauopathy

Sarah M Waldherr; Marina Han; Aleen D Saxton; Taylor A Vadset; Pamela J McMillan; Jeanna M Wheeler; Nicole F Liachko; Brian C Kraemer

doi:10.1038/s42003-024-06570-2

. 2024 Jul 25;7:903. doi: 10.1038/s42003-024-06570-2

Endoplasmic reticulum unfolded protein response transcriptional targets of XBP-1s mediate rescue from tauopathy

Sarah M Waldherr ^1,², Marina Han ^2,³, Aleen D Saxton ¹, Taylor A Vadset ³, Pamela J McMillan ⁴, Jeanna M Wheeler ¹, Nicole F Liachko ^1,^2,³, Brian C Kraemer ^1,^2,^3,^4,^5,^✉

PMCID: PMC11282107 PMID: 39060347

Abstract

Pathological tau disrupts protein homeostasis (proteostasis) within neurons in Alzheimer’s disease (AD) and related disorders. We previously showed constitutive activation of the endoplasmic reticulum unfolded protein response (UPR^ER) transcription factor XBP-1s rescues tauopathy-related proteostatic disruption in a tau transgenic Caenorhabditis elegans (C. elegans) model of human tauopathy. XBP-1s promotes clearance of pathological tau, and loss of function of the ATF-6 branch of the UPR^ER prevents XBP-1s rescue of tauopathy in C. elegans. We conducted transcriptomic analysis of tau transgenic and xbp-1s transgenic C. elegans and found 116 putative target genes significantly upregulated by constitutively active XBP-1s. Among these were five candidate XBP-1s target genes with human orthologs and a previously known association with ATF6 (csp-1, dnj-28, hsp-4, ckb-2, and lipl-3). We examined the functional involvement of these targets in XBP-1s-mediated tauopathy suppression and found loss of function in any one of these genes completely disrupts XBP-1s suppression of tauopathy. Further, we demonstrate upregulation of HSP-4, C. elegans BiP, partially rescues tauopathy independent of other changes in the transcriptional network. Understanding how the UPR^ER modulates pathological tau accumulation will inform neurodegenerative disease mechanisms and direct further study in mammalian systems with the long-term goal of identifying therapeutic targets in human tauopathies.

Subject terms: Alzheimer's disease, Prions, Behavioural genetics, Molecular neuroscience

Genomics and transcriptomics data evaluate XBP-1s target genes conferring protection from pathological tau protein in C. elegans.

Introduction

Proteins, which are the essential building blocks and catalytic machines of all cells, have a finite lifetime and can be prone to misfolding. Depending on the subcellular compartment, there are specific protein quality control mechanisms that respond to the presence of abnormal proteins, such as the cytoplasmic heat shock response, the endoplasmic reticulum (ER) unfolded protein response (UPR^ER) with subsequent ER-associated degradation (ERAD), and the mitochondrial unfolded protein response (UPR^mt). Once detected, multiple cytoplasmic quality control systems exist to degrade unnecessary or damaged proteins via proteolysis in order to maintain protein homeostasis (proteostasis), including the ubiquitin-proteasome system (UPS), chaperone-mediated autophagy (CMA), and macroautophagy, which will be referred to as autophagy¹.

Accumulation of unfolded proteins in the ER activates a transcriptional induction pathway known as the UPR^ER (Fig. 1a). Primary targets of the UPR^ER are molecular chaperones and folding enzymes localized in the ER; induction of these proteins augments the capacity of the protein folding system to restore ER proteostasis. In addition, some of the proteins involved in the ERAD system clear misfolded proteins from the ER and are upregulated by the UPR^ER. To understand transcriptional activation of the UPR^ER, previous work identified the cis-acting element and trans-acting factors responsible for the mammalian UPR^ER. The cis-acting ER stress response element (ERSE) with the consensus sequence CCAAT-N9-CCACG is necessary and sufficient for UPR^ER transcriptional induction^2,3. Because CCAAT of the ERSE is a binding site for the general transcription factor nuclear transcription factor Y (NF-Y)³, CCACG of the ERSE provides specificity to the UPR^ER in mammals⁴.

Mammalian inositol-requiring enzyme 1 α (IRE1α) is a type I ER-membrane bound endoribonuclease that initiates spliceosome-independent mRNA splicing of X-box binding protein 1 (XBP1) in response to ER stress⁵. The spliced form of XBP1 (XBP1s) functions as a potent transcription factor via frame switch splicing, joining the basic leucine zipper (bZIP) and transactivation domains⁴. XBP1s binds directly to the ERSE in collaboration with NF-Y to activate transcription of ER chaperone genes⁴.

Mammalian activating transcription factor 6 (ATF6) is a type II transmembrane protein in the ER activated by trans-compartmental proteolysis^6–9. In response to ER stress, ER membrane-bound ATF6 is converted into the active transcription factor ATF6n in the Golgi apparatus, and initiates transcription of ER chaperone genes via direct binding to the ERSE in collaboration with NF-Y. ATF6n can bind to the CCACG region of the ERSE as a homodimer or heterodimer only when NF-Y is bound to the CCAAT region^10,11.

The coordination of the three UPR^ER stress sensing and signaling pathways [protein kinase RNA-like ER kinase (PERK), IRE1α, and ATF6] in transcriptional remodeling has yet to be fully investigated. Previous studies indicate the activation of both the IRE1α/XBP1 and ATF6 pathways culminates in enhanced transcription at ERSE sites, thus upregulating the levels of ER chaperones¹². The co-dependence of the IRE1α and ATF6 branches merge at XBP1: XBP1 mRNA is induced by ATF6n and spliced by IRE1α⁴, and ATF6n heterodimerizes with XBP-1s to produce more potent transcriptional activation than XBP-1s homodimers¹³. The PERK pathway is mainly responsible for translational control, but it also plays a role in transcriptional control in mammals via induction of the transcription factor activating transcription factor 4 (ATF4). However, the ERSE is not a direct binding site of the transcription factor ATF4¹⁴.

Neurons are particularly vulnerable to challenges maintaining protein quality control over the lifetime. Post-mitotic neurons are more susceptible to the accumulation of cytotoxic proteins, since toxic substances cannot be diluted via cell division¹⁵. Throughout the course of aging, maintaining neuronal proteostasis becomes increasingly difficult. Components of the UPS, CMA, and autophagy in neurons are shown to be downregulated in both expression and activity¹⁶. Cytotoxicity and neuronal death resulting from misfolded oligomers and aggregates is a common molecular mechanism underlying the pathogenesis of many neurodegenerative diseases¹⁷.

Tau represents the most common misfolded protein in human neurodegenerative diseases¹⁸ and serves as a marker of brain aging. Tau proteinopathies (tauopathies) are a heterogeneous group of diseases characterized by accumulation of abnormal cytoplasmic hyperphosphorylated tau protein and associated with cognitive and motor impairments¹⁹. These diseases include familial frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD-tau), argyrophilic grain disease (AGD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), Pick’s disease (PiD), and Alzheimer’s disease (AD)²⁰. AD is the most common tauopathy and cause of dementia, and its neuropathology also includes extracellular amyloid plaques composed of fibrillar amyloid beta (Aβ) peptides²¹. Mutations in the microtubule-associated protein tau (MAPT) gene encoding the protein tau cause FTDP-17, providing a direct link between tau dysfunction and neurodegenerative disease^22–24. Thus far, no interventions have been found to reduce neurodegeneration or tau accumulation in these diseases.

UPR^ER activation has been found in postmortem brain tissue of patients with tauopathies²³. The molecular chaperone binding immunoglobulin protein (BiP), phosphorylated PERK (pPERK), eukaryotic initiation factor 2 α (eIF2α), and IRE1α are upregulated in hippocampal neurons in AD^25,26, while pPERK and phosphorylated IRE1α are increased in PSP, PiD, and FTLD-tau^27,28. Of note, UPR^ER markers were present in neurons with diffuse phosphorylated tau, but rarely in neurons without phosphorylated tau or with aggregated phosphorylated tau, suggesting that UPR^ER activation occurs early on in tauopathy pathogenesis^26–28. A recent study of late-stage AD, PiD, and PSP found no significant difference in XBP-1s, phosphorylated eIF2α, BiP, or CCAAT-enhancer-binding-protein homologous protein (CHOP) compared to non-demented controls²⁹, further supporting the notion that UPR^ER activation is involved in the early stages of tauopathy.

Given the significance of the UPR^ER in aging and neurodegenerative diseases, our previous work studied genetic manipulation of the UPR^ER in pan-neuronal human wild type tau transgenic C. elegans models³⁰. First, we used mild models of human wild type tau toxicity [Tau (low) transgenic C. elegans models]^30,31 and found loss of function of XBP-1 and ATF-6 UPR^ER transcriptional branches enhanced tauopathy phenotypes, including exacerbated behavior defects, increased tau protein accumulation, and more severe neurodegeneration³⁰. Next, we investigated the effects of UPR^ER activation in the absence of ER stress using the pan-neuronal constitutively active xbp-1s transgenic C. elegans model (xbp-1s Tg)³² and a severe model of wild type tau toxicity [Tau (high) transgenic C. elegans model]^30,31. XBP-1s gain of function ameliorated tauopathy phenotypes in Tau (high) transgenic C. elegans, including rescued behavior defects, reduced tau accumulation, and decreased neurodegeneration, which was dependent on functional ATF-6³⁰. Additionally, we showed functional ERAD via sel-11 is required for XBP-1s-mediated tauopathy suppression in C. elegans³⁰. Because the two main transcriptional branches of the UPR^ER initiate downstream activation of genes involved in ERAD, here we conducted transcriptomic analysis of XBP-1s target genes in C. elegans models of tauopathy. We also used putative loss-of-function mutations and gain-of-function transgenes to genetically dissect the individual roles of each target gene in XBP-1s-mediated transcriptional remodeling in tau proteostasis. Finally, we explored the translational capability of targeting the XBP1s transcriptional branch of the UPR^ER in mammals by generating a conditional neuronal XBP1s (niXBP1s) overexpression mouse model.

Results

Transcriptomic analysis reveals candidate XBP-1s target genes involved in tauopathy suppression in C. elegans

The IRE1α/XBP1 pathway is the most conserved branch of the UPR^ER, leading to the expression of the master UPR^ER transcription factor XBP1s^33,34. To probe UPR^ER function in C. elegans neurons, we utilized pan-neuronal xbp-1s gain-of-function transgenic animals (xbp1-s Tg) driving transcriptional activation of the XBP-1s UPR^ER branch in the absence of ER stress³². In our previous work, we demonstrated XBP-1s overexpression in neurons can rescue tauopathy in a C. elegans model of human tauopathy³⁰. Because XBP-1s is a bZIP type transcription factor, we hypothesized XBP-1s transcriptional changes may contribute to tauopathy amelioration in C. elegans. To test this, we again used the pan-neuronal Tau (high) transgenic C. elegans model [Tau (high)], which exhibits severe behavioral phenotypes, accumulation of pathological tau protein, and neurodegeneration^30,31. We previously demonstrated tauopathy rescue by xbp-1s Tg overexpression is not mediated by tau mRNA reduction, but rather changes in tau protein accumulation^30,35. Transcriptomic analysis was conducted on young C. elegans at the L2 larval stage. Using the L2 larval stage as our time point for analysis provides two main advantages. First, neurodegeneration in tau transgenic animals has yet to begin³⁶. Second, the full complement of neurons is present at the L2 larval stage, but other cell types such as germ cells and somatic gonad remain as progenitor cells, yielding the highest ratio of neurons to other cell types in the animal at any developmental stage³⁷. We sequenced RNA from large, synchronized L2 larval stage populations of non-transgenic (non-Tg), xbp-1s Tg, Tau (high), and Tau (high); xbp-1s Tg animals using Illumina based sequencing technology. Transcriptomic analysis of the mRNA populations revealed 116 genes with robust and statistically significant expression changes (i.e., at least two-fold change in xbp-1s Tg versus non-Tg) (Fig. 1b–e). These candidate XBP-1s target genes for mediating tauopathy suppression are shown in Supplementary Table 1 and served as the basis for identifying regulators of tauopathy. As a control, we also analyzed tau transgene mRNA abundance and showed xbp-1s Tg does not alter accumulation of tau message (Supplementary Table 2), consistent with previously published observations^30,35. Gene Ontology enrichment analysis for significantly overrepresented target genes is presented in Fig. 1f.

The three UPR^ER branches exert unique and coordinated transcriptional and translational remodeling to restore proteostasis, with the IRE1α/XBP1 and ATF6 branches controlling the majority of transcriptional changes. In C. elegans, our previous work revealed xbp-1s-mediated tauopathy suppression requires a functional ATF-6 branch³⁰, and other studies suggest the ATF-6n transcriptional pathway might have evolved as a backup mechanism to the XBP-1s transcriptional pathway³⁸. In mammals, ATF6n and XBP1s can form transcriptionally active heterodimers, which exhibit a higher binding affinity to target genes than XBP1s homodimers^13,39. Therefore, we hypothesized the gene(s) responsible for rescue of tauopathy in C. elegans might be responsive to both XBP-1s and ATF-6n. To examine this possibility, we first analyzed the candidate XBP-1s target genes identified in Supplementary Table 1 for conservation between C. elegans and humans, revealing 11 genes (Table 1). Next, we investigated the conserved XBP1s target genes for literature evidence of ATF6n responsiveness (Table 2). From this analysis, we identified five genes exhibiting significant responsiveness to XBP-1s in the transcriptomic data that also have a previously demonstrated functional gene classification association with ATF6n: caspase 1 (csp-1; homologous to caspase gene family, such as CASP3, 6, 7, and 14)⁴⁰, DNAJ domain (prokaryotic heat shock protein) 28 (dnj-28; homologous to DNAJC3/ p58^IPK)⁴¹, heat shock protein 4 (hsp-4; homologous to HSP5A/BiP/GRP-78)^9,42, choline kinase beta 2 (ckb-2; homologous to CHKA and CHKB)⁴³, and lipase like 3 (lipl-3; homologous to lipase gene family, such as LIPA, LIPF, LIPJ, LIPK, LIPM, and LIPN)⁴⁴.

Table 1.

XBP-1s responsive genes identified by RNAseq in C. elegans with human orthologs

Worm gene	Human gene	Protein description (subcellular localization)	RNA fold change (xbp-1s Tg vs. non-Tg)
lipl-3	LIP family	Lipase (Lysosome)	18.205 ↑
csp-1	CASP family	Aspartyl Protease (Nucleus)	10.596 ↑
dnj-28	DNAJC3	DNAJ, co-chaperone of HSP70 (ER)	6.711 ↑
F41E7.6	CROT	Carnitine O-Octanoyltransferase (Peroxisome)	4.315 ↑
erp-44.3	ERP44	Protein Disulfide Isomerase (ER)	3.980 ↑
C01B4.6	GALM	Galactose Mutarotase (Cytoplasm)	3.394 ↑
Y19D10A.16	GALM	Galactose Mutarotase (Cytoplasm)	3.061 ↑
mct-2	SLC16A14	Carnitine Transporter (Plasma Membrane)	2.917 ↑
hsp-4	HSPA5/BiP/GRP-78	HSP70, chaperone (ER)	2.911 ↑
ckb-2	CHKA, CHKB	Choline Kinase (Cytoplasm)	2.151 ↑
eol-1	DXO	Decapping Exoribonuclease (Nucleus)	2.034 ↑

Open in a new tab

Table 2.

XBP-1s responsive genes identified by RNAseq in C. elegans with human orthologs and ATF6 association

Worm gene	Human gene	Protein description (subcellular localization)	RNA fold change (xbp-1s Tg vs. non-Tg)	ATF6 association
lipl-3	LIP family	Lipase (Lysosome)	18.205 ↑	⁴⁴
csp-1	CASP family	Aspartyl Protease (Nucleus)	10.596 ↑	⁴⁰
dnj-28	DNAJC3	DNAJ, Co-chaperone of HSP70 (ER)	6.711 ↑	⁴¹
hsp-4	HSPA5/BiP/GRP-78	HSP70, chaperone (ER)	2.911 ↑	^9,42
ckb-2	CHKA, CHKB	Choline Kinase (Cytoplasm)	2.151 ↑	⁴³

Open in a new tab

A suite of ATF6-dependent XBP-1s target genes is required for C. elegans tauopathy suppression

To validate the transcriptional analysis of XBP-1s responsive genes in C. elegans with a known association with ATF6 (Table 2), we examined available csp-1, dnj-28, hsp-4, ckb-2, and lipl-3 putative loss-of-function mutant strains (Supplementary Table 3) in the Tau (high); xbp-1s Tg C. elegans background. We hypothesized if these five target genes are necessary, genetic loss of function might alter xbp-1s-mediated tauopathy suppression in C. elegans. Using the same strategy employed characterizing the UPR^ER in C. elegans tauopathy³⁰, we first analyzed locomotion behavior, which provides a sensitive readout of neuronal function^{30,31,36,45–59}. If the target gene appeared necessary for xbp-1s-mediated rescue of tau-induced behavioral defects, we also measured changes in total tau protein levels.

The csp-1 gene in C. elegans is orthologous to the cysteine-aspartic acid protease (caspase; CASP) family of genes in humans, such as CASP3, CASP6, CASP7, and CASP14⁶⁰. Caspases are protease enzymes playing essential roles in programmed cell death, or apoptosis. The mechanism of apoptosis is evolutionarily conserved, and the critical involvement of caspase cell death abnormality 3 (CED-3) protein in apoptosis was first discovered in C. elegans⁶¹. In mammals, 14 caspases have currently been identified and are functionally divided into three subfamilies: apoptosis initiator (CASP2, CASP8, CASP9, CASP10), apoptosis effector (CASP3, CASP6, CASP7), and inflammatory mediator (CASP1, CASP4, CASP5, CASP11, CASP12, CASP13, CASP14)^62,63.

The consequence of caspase activity loss of function in xbp-1s-mediated tauopathy suppression was analyzed using a C. elegans mutant strain [csp-1 (ok2570); referred to as csp-1 (−/−)]. When compared to non-Tg animals, csp-1 loss of function modestly enhances locomotion (Supplementary Fig. 1). We previously showed xbp-1s overexpression modestly induces locomotion defects when compared to non-Tg animals³⁰. Tau (high); xbp-1s animals were crossed with csp-1 (−/−) animals to understand the effect on tauopathy phenotypes. When we examined the deletion of csp-1 in Tau (high) animals, we found there was no change in tau-induced behavioral dysfunction when compared to Tau (high) animals alone (Fig. 2a). As we have shown previously³⁰, Tau (high); xbp-1s Tg animals exhibited robust rescue of the tau-mediated locomotion defects compared to Tau (high) animals alone (Fig. 2a), with a 57% rescue in motility defects compared to non-Tg^30,35. Interestingly, csp-1 (−/−) loss of function abolished the ability of xbp-1s to suppress locomotion defects as seen in Tau (high); xbp-1s Tg; csp-1 (−/−) animals compared to Tau (high); xbp-1s Tg animals (Fig. 2a), which suggests csp-1 upregulation by xbp-1s is necessary for xbp-1s-mediated tauopathy suppression.

Fig. 2 — Shown are genetic analyses of XBP-1s/ATF6 targets on tauopathy phenotypes in transgenic *C. elegans*. a *csp-1* loss of function abolishes the ability of neuronal overexpression of *xbp-1s* in Tau (high) animals to suppress severe behavioral defects observed as reflexive locomotion in response to a liquid environment [n = 75 animals; N = 5 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (****p ≤ 0.0001)]. b, c *csp-1* loss of function abolishes the ability of neuronal overexpression of *xbp-1s* in Tau (high) animals to decrease soluble tau protein levels. Representative immunoblots for total tau (DAKO pAb) and tubulin are shown, and densitometry analysis of chemiluminescence signals for tau normalized to tubulin are plotted [N = 4 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (***p ≤ 0.001, ****p ≤ 0.0001)]. d *ced-3* loss of function abolishes the ability of neuronal overexpression of *xbp-1s* in Tau (high) animals to suppress severe behavioral defects observed as reflexive motor impairment in response to a liquid environment [n = 75 animals; N = 5 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (****p ≤ 0.0001)]. e, f *ced-3* loss of function abolishes the ability of neuronal overexpression of *xbp-1s* in Tau (high) animals to decrease soluble tau protein levels. Representative immunoblots for total tau (SP70 pAb) and tubulin are shown, and densitometry analysis of chemiluminescence signals for tau normalized to tubulin are plotted [N = 5 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (**p ≤ 0.01, ***p ≤ 0.001)]. All bar graphs represent mean + SEM. Arrowhead on y-axis of all liquid thrashing bar graphs denotes non-Tg animals average ~70 thrashes/min under standard laboratory conditions. Source data are available as Supplmentary Data.

Because xbp-1s-mediated tauopathy behavioral suppression requires csp-1, we also measured csp-1 loss of function effects on total tau protein by immunoblot. Loss of function of csp-1 did not change tau protein levels in Tau (high) animals compared to Tau (high) animals alone (Fig. 2b, c). As shown previously³⁰, xbp-1s overexpression reduced tau protein accumulation in the Tau (high) Tg background (Fig. 2b, c), without affecting human MAPT transcript levels³⁰ (Supplementary Table 2). Interestingly, Tau (high); xbp-1s Tg; csp-1 (−/−) animals accumulated total tau protein comparable to the level seen in Tau (high) animals (Fig. 2b, c).

In C. elegans, there are four caspase genes (ced-3, csp-1, csp-2, and csp-3), and csp-1 promotes programmed cell death in parallel to the canonical apoptosis pathway involving CED-3 activation⁶⁴. To test whether the involvement of csp-1 in xbp-1s-mediated tauopathy suppression is broadly applicable to overall caspase function, we crossed ced-3 (−/−) animals [ced-3 (n1286)] with Tau (high); xbp-1s animals. Behaviorally, ced-3 loss of function causes locomotion defects when compared to non-Tg animals (Supplementary Fig. 1). In the Tau (high) background, ced-3 loss of function does not affect movement defects (Fig. 2d). Surprisingly, Tau (high); xbp-1s Tg; ced-3 (−/−) animals also exhibited movement defects similar to Tau (high) animals alone (Fig. 2d). The mild locomotion defects evident in ced-3 (−/−) animals may partially block tauopathy suppression. However, we would expect the change in phenotype to be proportional to the defects in ced-3 (−/−) animals alone instead of the complete elimination of suppression that was observed, which suggests ced-3 is also necessary for xbp-1s-mediated tauopathy suppression.

Because xbp-1s-mediated tauopathy behavioral suppression requires ced-3, we also measured ced-3 loss of function effects on total tau protein by immunoblot. Loss of function of ced-3 in the Tau (high) background did not alter tau protein levels when compared to Tau (high) animals alone (Fig. 2e, f). Interestingly, Tau (high); xbp-1s Tg; ced-3 (−/−) animals accumulated total tau protein comparable to the level seen in Tau (high) animals (Fig. 2e, f). Taken together, these data demonstrate overall caspase activity via either CSP-1 or CED-3 is necessary for xbp-1s gain of function to suppress behavioral and biochemical tauopathy phenotypes observed in the C. elegans Tau (high) background.

C. elegans dnj-28 is orthologous to human DNAJC3, which encodes the ER stress-regulated chaperone DNAJ heat shock protein family (Hsp40) member C3 (DNAJC3), also known as 58-kilodalton inhibitor of protein kinase (P58^IPK). ER stress induces the transcription of DNAJC3⁶⁵. DNAJC3 has diverse functions in the ER, including being a co-chaperone and regulator of BiP^66–69 as well as inhibiting PERK, thereby relieving the PERK-mediated translational attenuation⁶⁵.

We investigated the effects of dnj-28 loss of function in xbp-1s-mediated tauopathy suppression in C. elegans by using a putative loss-of-function mutant strain [dnj-28 (ok2490); referred to as dnj-28 (−/−) A] crossed to Tau (high); xbp-1s Tg animals. Compared to non-Tg animals, there is no significant difference in movement with dnj-28 loss of function (Supplementary Fig. 2). Deletion of dnj-28 in Tau (high) animals did not change the tau-induced locomotion abnormalities when compared to Tau (high) animals alone (Fig. 3a). However, dnj-28 loss of function prevented the ability of xbp-1s Tg to suppress behavioral defects in Tau (high); xbp-1s Tg; dnj-28 (−/−) A animals when compared to Tau (high); xbp-1s Tg animals (Fig. 3a). To validate these findings, we generated an additional independent molecular null allele in dnj-28 where the entire coding sequence of dnj-28 was deleted [dnj-28 (bk3074); referred to as dnj-28 (−/−) B]. Analysis of dnj-28 (−/−) B replicated the findings in dnj-28 (−/−) A (Supplementary Fig. 3).

Fig. 3 — Shown are genetic analysis of XBP-1s/ATF6n targets on tauopathy phenotypes in transgenic *C. elegans*. a *dnj-28* loss of function abolishes the ability of neuronal overexpression of *xbp-1s* in Tau (high) animals to suppress severe behavioral defects observed as reflexive motor impairment in response to a liquid environment [n = 97 animals; N = 7 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (****p ≤ 0.0001)]. b, c *dnj-28* loss of function trends toward but does not significantly reduce soluble tau protein levels caused by neuronal *xbp-1s* overexpression in Tau (high) animals. Representative immunoblots for total tau (DAKO pAb) and tubulin are shown, and densitometry analysis of chemiluminescence signals for tau normalized to tubulin are plotted [N = 3 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (**p ≤ 0.01)]. d *dnj-27* loss of function abolishes the ability of neuronal overexpression of *xbp-1s* in Tau (high) animals to suppress severe behavioral defects observed as reflexive motor impairment in response to a liquid environment [n = 80, 74, 80, 80 animals, respectively; N = 4 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (****p ≤ 0.0001)]. e *dnj-28* loss of function in Tau (low) animals does not alter mild behavioral defects observed as reflexive motor impairment in response to a liquid environment [n = 96 and 94 animals, respectively; N = 5 biologically independent experiments; statistical analysis is by unpaired t-test, two tailed (ns: p = 0.2586)]. All bar graphs represent mean + SEM. Arrowhead on y-axis of all liquid thrashing bar graphs denotes non-Tg animals average ~70 thrashes/min under standard laboratory conditions. Source data are available as Supplmentary Data.

Because dnj-28 is required for xbp-1s-mediated tauopathy behavioral suppression, we also measured the effects of dnj-28 loss of function on total tau protein by immunoblot. Loss of function of dnj-28 alone did not change tau protein levels in Tau (high) animals compared to Tau (high) animals alone (Fig. 3b, c). Interestingly, Tau (high); xbp-1s Tg; dnj-28 (−/−) A animals accumulated total tau protein comparable to the level seen in Tau (high) animals (Fig. 3b, c).

The DNAJ domain class of genes in C. elegans includes 29 genes⁶⁰. To test whether the requirement of functional dnj-28 for xbp-1s-mediated tauopathy suppression is broadly applicable to other DNAJ domain genes, we chose to investigate dnj-27 loss of function [dnj-27 (ok2302); referred to as dnj-27 (−/−)]. Downregulation of dnj-27 expression by RNAi in human Aβ, α-synuclein, and polyglutamine (polyQ) transgenic C. elegans models enhances neurodegenerative phenotypes⁷⁰. In the Tau (high) background, dnj-27 loss of function does not affect movement defects (Fig. 3d). Surprisingly, Tau (high); xbp-1s Tg; dnj-27 (−/–) animals also exhibited movement defects similar to Tau (high) alone animals (Fig. 3d). Therefore, two DNAJ domain genes (dnj-28 and dnj-27) are both required for xbp-1s-mediated tauopathy suppression in C. elegans.

In addition, we probed the importance of dnj-28 in modulating tauopathy. Because the severity of behavioral dysfunction of Tau (high) animals might affect our ability to discriminate enhancement of tauopathy phenotypes, we used the pan-neuronal Tau (low) transgenic C. elegans model, which exhibits mild behavioral deficits and no significant accumulation of pathological tau species^30,31. If dnj-28 regulates tau proteostasis in C. elegans, we expected Tau (low) animals crossed with the dnj-28 loss-of-function mutant strain would exhibit enhancement of tau-dependent locomotion dysfunction. However, when crossed with Tau (low) animals, dnj-28 (−/−) A did not worsen Tau (low) behavioral defects (Fig. 3e), demonstrating that dnj-28 loss of function does not exacerbate tauopathy in the absence of overt UPR^ER induction by xbp-1s overexpression.

The C. elegans gene hsp-4 is an ortholog of the human gene heat shock protein A5 (HSPA5), which encodes the UPR^ER sensor protein BiP. As one of the most abundant proteins in the ER, BiP maintains ER homeostasis via a variety of functions, including protein folding processes, protein import into the ER, regulation of calcium homeostasis, and facilitation of ERAD⁷¹. Under ER stress conditions, BiP can initiate the UPR^ER, decrease unfolded protein load, induce autophagy, and crosstalk with apoptosis machinery to assist in the cell survival decision⁷¹.

To understand the potential role of BiP in xbp-1s-mediated tauopathy suppression in C. elegans, we used an hsp-4 loss-of-function mutant strain [hsp-4 (gk514); referred to as hsp-4 (−/−) A]. When compared to non-Tg animals, hsp-4 (−/−) A animals had mild locomotion defects (Supplementary Fig. 4). Deletion of hsp-4 in Tau (high) animals did not affect tau-induced behavioral dysfunction when compared to Tau (high) animals alone (Fig. 4a). However, hsp-4 loss of function completely eliminated xbp-1s-mediated rescue of behavioral phenotype in Tau (high); xbp-1s Tg; hsp-4 (−/−) A animals (Fig. 4a). The mild locomotion defects evident in hsp-4 (−/−) A animals may partially block tauopathy suppression. Although we would expect the change in phenotype to be proportional to the defects in hsp-4 (−/−) A animals alone instead of the complete reversal of suppression that was observed, which suggests hsp-4 upregulation by xbp-1s is necessary for xbp-1s-mediated tauopathy suppression. To validate these findings, we generated an additional independent molecular null allele in hsp-4 where the entire coding sequence of hsp-4 was deleted [hsp-4 (bk3060); referred to as hsp-4 (−/−) B]. Analysis of hsp-4 (−/−) B replicated the findings in hsp-4 (−/−) A (Supplementary Fig. 5).

Fig. 4 — Shown are genetic analyses of XBP-1s/ATF6 targets on tauopathy phenotypes in transgenic *C. elegans*. a *hsp-4* loss of function abolishes the ability of neuronal overexpression of *xbp-1s* in Tau (high) animals to suppress severe behavioral defects observed as reflexive motor impairment in response to a liquid environment [n = 90 animals; N = 6 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (****p ≤ 0.0001)]. b, c *hsp-4* loss of function abolishes the ability of neuronal overexpression of *xbp-1s* in Tau (high) animals to decrease soluble tau protein levels. Representative immunoblots for total tau (SP70 pAb) and tubulin are shown, and densitometry analysis of chemiluminescence signals for tau normalized to tubulin are plotted [N = 3 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (**p ≤ 0.01, ***p ≤ 0.001)]. d *hsp-3* loss of function does not alter the ability of neuronal overexpression of *xbp-1s* in Tau (high) animals to suppress severe behavioral defects observed as reflexive motor impairment in response to a liquid environment [n = 60 animals; N = 4 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (****p ≤ 0.0001)]. e *hsp-4* loss of function in Tau (low) animals enhances mild behavioral defects observed as reflexive motor impairment in response to a liquid environment [n = 100 animals; N = 5 biologically independent experiments; statistical analysis is by unpaired t-test, two tailed (****p ≤ 0.0001)]. f, g *hsp-4* loss of function in Tau (low) animals does not affect soluble tau protein levels. Representative immunoblots for total tau (SP70 pAb) and tubulin are shown, and densitometry analysis of chemiluminescence signals for tau normalized to tubulin are plotted [N = 4 biologically independent experiments; statistical analysis is by unpaired t-test, two-tailed (ns: p = 0.9003)]. h Neuronal high overexpression of *hsp-4* in Tau (high) animals does not alter severe behavioral defects, while neuronal low overexpression of *hsp-4* in Tau (high) animals mildly suppresses severe behavioral defects observed as reflexive motor impairment in response to a liquid environment [n = 70, 70, and 69 animals, respectively; N = 5 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (****p ≤ 0.0001)]. i Neuronal high overexpression of *hsp-4* in Tau (high) animals enhances severe behavioral defects, while neuronal low overexpression of *hsp-4* in Tau (high) animals mildly suppresses severe behavioral defects observed in an unstimulated environment [n = 60 animals; N = 3 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (****p ≤ 0.0001)]. j, k Neuronal high overexpression of *hsp-4* in Tau (high) animals increases soluble tau protein levels, while neuronal low overexpression of *hsp-4* in Tau (high) animals does not affect soluble tau protein levels. Representative immunoblots for total tau (DAKO pAb) and tubulin are shown, and densitometry analysis of chemiluminescence signals for tau normalized to tubulin are plotted [N = 4 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (***p ≤ 0.001)]. All bar graphs represent mean + SEM. Arrowhead on y-axis of all liquid thrashing bar graphs denotes non-Tg animals average ~70 thrashes/min under standard laboratory conditions. Source data are available as Supplmentary Data.

Because xbp-1s-mediated tauopathy behavioral suppression requires hsp-4, we also measured hsp-4 loss of function effects on total tau protein by immunoblot. Loss of function of hsp-4 alone did not change tau protein levels in Tau (high) animals compared to Tau (high) animals alone (Fig. 4b, c). Interestingly, Tau (high); xbp-1s Tg; hsp-4 (−/−) A animals accumulated total tau protein comparable to the level seen in Tau (high) animals (Fig. 4b, c). Taken together, these data demonstrate hsp-4 loss of function significantly impacts the ability of xbp-1s gain of function to suppress behavioral and biochemical tauopathy phenotypes observed in the C. elegans Tau (high) background.

In C. elegans, HSP-3 and HSP-4 are homologous to mammalian BiP. HSP-3 is both constitutively expressed and stress-responsive, while HSP-4 has very low basal expression in most cells, but is strongly induced by UPR^ER signaling^72,73. Although hsp-4 was responsive to xbp-1s upregulation in our transcriptomics data, hsp-3 was not. Tau (high); xbp-1s Tg animals were crossed with a C. elegans hsp-3 mutant strain [hsp-3 (ok1083); referred to as hsp-3 (−/−)]. When compared to non-Tg animals, hsp-3 (−/−) animals had mild locomotion defects (Supplementary Fig. 2). Tau (high); hsp-3 (−/−) animals displayed a similar defect in locomotion when compared to Tau (high) animals alone (Fig. 4d). When compared to Tau (high); xbp-1s Tg animals, Tau (high); xbp-1s Tg; hsp-3 (−/−) animals exhibited similar suppression of tauopathy-induced locomotion defects (Fig. 4d), indicating hsp-3 loss of function is dispensable for xbp-1s-mediated tauopathy suppression. Although HSP-3 and HSP-4 proteins are highly conserved and thought to be functionally redundant⁷³, we show xbp-1s-mediated tauopathy suppression in C. elegans is specific, requiring functional HSP-4.

Given the lack of hsp-3 involvement, we further explored the impact of HSP-4 function on modulating tauopathy phenotypes in the absence of overt UPR^ER induction by xbp-1s overexpression in neurons. Because of the severity of behavioral dysfunction of Tau (high) animals, we again used Tau (low) animals. We observed Tau (low); hsp-4 (−/−) A animals displayed significant enhancement of the locomotion defects seen in Tau (low) Tg animals alone (Fig. 4e). We further probed hsp-4 involvement in tau proteostasis and found no significant increase in total tau species detected by immunoblot in Tau (low); hsp-4 (−/−) A animals compared to Tau (low) animals (Fig. 4f, g). Additionally, we observed no change in phosphorylated tau in Tau (low); hsp-4 (−/−) animals (Supplementary Fig. 6).

Because HSP-4 loss of function blocks xbp-1s-mediated tauopathy suppression of behavioral and biochemical phenotypes in the Tau (high) background and enhances tauopathy phenotypes in the Tau (low) background, we hypothesized hsp-4 gain of function alone might play a role in tauopathy in C. elegans. We generated pan-neuronal hsp-4 transgenic C. elegans lines with varying expression levels (Supplementary Fig. 7) using a different neuronal promoter than those driving Tau (high) and xbp-1s Tg C. elegans expression. Both neuronal low overexpression [hsp-4 (low) Tg] and high overexpression [hsp-4 (high) Tg] C. elegans models were generated and have limited or no impact on health and motor function (Supplementary Figs. 8 and 9). We crossed hsp-4 Tg animals with Tau (high) animals and analyzed the effect on tau-induced locomotion defects. Interestingly, when compared to Tau (high) animals alone, Tau (high); hsp-4 (high) Tg animals exhibited similar tau-induced behavioral defects as Tau (high) animals (Fig. 4h). However, low overexpression of hsp-4 was able to mildly suppress behavioral tauopathy phenotypes, as seen in Tau (high); hsp-4 (low) Tg animals compared to Tau (high) animals alone (Fig. 4h). To confirm the effect of hsp-4 overexpression levels on tauopathy movement phenotypes, we used the radial locomotion assay, which measures movement in the absence of an external stimulus. Interestingly, there was a significant reduction in radial dispersion of Tau (high); hsp-4 (high) Tg animals compared to Tau (high) animals alone, while hsp-4 low overexpression modestly increased radial dispersion in the Tau (high) background (Fig. 4i). This confirms our other behavioral results for hsp-4 low overexpression, with this assay also able to discriminate the movement defect with hsp-4 high overexpression in the Tau (high) background (Fig. 4h). We also examined the consequence of hsp-4 overexpression on tau proteostasis by immunoblot and found high overexpression of hsp-4 in the Tau (high) background significantly increases total tau species (Fig. 4j, k). However, low overexpression of hsp-4 in the Tau (high) background does not significantly alter total tau protein levels when compared to Tau (high) animals (Fig. 4j, k). These results indicate HSP-4 function can mediate tauopathy suppression in C. elegans, but requires a fine-tuned expression level.

The C. elegans gene ckb-2 is an ortholog of human choline kinase alpha (CHKA) and choline kinase beta (CHKB). These proteins participate in the cytidine diphosphocholine (CDP-choline) pathway to synthesize phosphatidylcholine for ER biogenesis, which is upregulated by both ATF6α⁴³ and XBP-1s⁷⁴. The ckb-2 loss of function [ckb-2 (bk3106); referred to as ckb-2 (−/−)] in Tau (high) animals did not change tau-induced motor deficits. However, deletion of ckb-2 in Tau (high); xbp-1s Tg animals eliminated the ability of xbp-1s to suppress tauopathy, indicating intact ckb-2 is required for xbp-1s to exert its suppressive effect (Fig. 5a).

Fig. 5 — Shown are genetic analyses of XBP-1s/ATF6 targets on tauopathy phenotypes in transgenic *C. elegans*. a *ckb-2* loss of function abolishes the ability of neuronal overexpression of *xbp-1s* in Tau (high) animals to suppress severe behavioral defects observed as reflexive motor impairment in response to a liquid environment [n = 80 animals; N = 4 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (****p ≤ 0.0001)]. b *lipl-3* loss of function abolishes the ability of neuronal overexpression of *xbp-1s* in Tau (high) animals to suppress severe behavioral defects observed as reflexive motor impairment in response to a liquid environment [n = 90 animals; N = 6 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (****p ≤ 0.0001)]. All bar graphs represent mean + SEM. Arrowhead on y-axis of all liquid thrashing bar graphs denotes non-Tg animals average ~70 thrashes/min under standard laboratory conditions. Source data are available as Supplmentary Data.

The lipl-3 gene in C. elegans is orthologous to a human class of genes known as lipase (LIP) family members, including LIPA, LIPF, LIPJ, LIPK, LIPM, and LIPN⁶⁰. The LIPA gene exhibits the highest homology to lipl-3 and encodes the lysosomal acid lipase (LAL) enzyme found in lysosomal compartments, which is essential for lipid metabolism. LAL breaks down lipids such as cholesterol esters and triglycerides to generate free fatty acids and cholesterol⁷⁵. To understand whether functional LIPL-3 is necessary for xbp-1s-mediated tauopathy suppression, we crossed Tau (high); xbp-1s Tg animals with a loss-of-function mutant strain [lipl-3 (gk846191); referred to as lipl-3 (−/−)] and explored the effect on tauopathy locomotion defects. Compared to non-Tg animals, there is no significant difference in movement with lipl-3 loss of function (Supplementary Fig. 2). Loss of function of lipl-3 did not significantly affect Tau (high) locomotion defects (Fig. 5b). In contrast, Tau (high); xbp-1s Tg; lipl-3 (−/−) animals displayed similar locomotion abnormalities when compared to Tau (high) animals alone (Fig. 5b), indicating lipl-3 is also required for xbp-1s-mediated tauopathy suppression in C. elegans.

Taken together, five genes identified as responsive to XBP-1s in the transcriptomic data with a previously known association with ATF6n (csp-1, dnj-28, hsp-4, ckb-2, and lipl-3; Table 2) are all essential for xbp-1s-mediated tauopathy suppression in C. elegans. However, in absence of overt UPR^ER induction by xbp-1s overexpression in neurons, these target genes differentially affect tauopathy phenotypes, which warrants future investigation.

A suite of other XBP-1s target genes is also required for C. elegans tauopathy suppression

To understand whether XBP-1s responsive genes in C. elegans rely on an association with ATF6 (Table 2), we also examined the remaining six C. elegans genes with human homologs, but no previous association with ATF6 (Table 1). We utilized available erp-44.3, F41E7.6, C01B4.6, Y19D10A.16, eol-1, and mct-2 putative loss-of-function mutant strains (Supplementary Table 3) in the Tau (high); xbp-1s Tg C. elegans background. We hypothesized if these target genes are also necessary for tauopathy suppression, their genetic loss of function would also block xbp-1s-mediated tauopathy suppression in C. elegans.

The C. elegans gene erp-44.3 is orthologous to human endoplasmic reticulum protein 44 (ERP44), which encodes a pH-regulated chaperone in the protein disulfide isomerase (PDI) family. ERP44 retrieves mislocalized or incompletely assembled proteins from the ER-Golgi intermediate compartment or cis-Golgi and deposits them back in the ER, thereby acting as a quality control mechanism for secreted proteins⁷⁶. Compared to non-Tg animals, there is no significant difference in movement with erp-44.3 loss of function [erp-44.3 (tm6492); referred to as erp-44.3 (−/−)] (Supplementary Fig. 2). By crossing erp-44.3 (−/−) into the Tau (high); xbp-1s Tg background, we showed loss of function of erp-44.3 blocked xbp-1s-mediated suppression of tau-induced locomotion deficits, while erp-44.3 loss of function in the Tau (high) background did not change locomotion compared to Tau (high) animals alone (Fig. 6a), indicating erp-44.3 is also required for xbp-1s-mediated tauopathy suppression.

Fig. 6 — Shown are genetic analyses of XBP-1s targets with human homologs lacking a known association with ATF6 on tauopathy phenotypes in transgenic *C. elegans*. a *erp-44.3* loss of function abolishes the ability of neuronal overexpression of *xbp-1s* in Tau (high) animals to suppress severe behavioral defects observed as reflexive motor impairment in response to a liquid environment [n = 75 animals; N = 5 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (****p ≤ 0.0001)]. b *F41E7.6* loss of function abolishes the ability of neuronal overexpression of *xbp-1s* in Tau (high) animals to suppress severe behavioral defects observed as reflexive motor impairment in response to a liquid environment [n = 75 animals; N = 5 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (****p ≤ 0.0001)]. c *C01B4.6* loss of function abolishes the ability of neuronal overexpression of *xbp-1s* in Tau (high) animals to suppress severe behavioral defects observed as reflexive motor impairment in response to a liquid environment [n = 75 animals; N = 5 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (****p ≤ 0.0001)]. d *Y19D10A.16* loss of function abolishes the ability of neuronal overexpression of *xbp-1s* in Tau (high) animals to suppress severe behavioral defects observed as reflexive motor impairment in response to a liquid environment [n = 70 animals; N = 5 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (****p ≤ 0.0001)]. e *eol-1* loss of function abolishes the ability of neuronal overexpression of *xbp-1s* in Tau (high) animals to suppress severe behavioral defects observed as reflexive motor impairment in response to a liquid environment [n = 80 animals; N = 4 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (****p ≤ 0.0001)]. f *mct-2* loss of function abolishes the ability of neuronal overexpression of *xbp-1s* in Tau (high) animals to suppress severe behavioral defects observed as reflexive motor impairment in response to a liquid environment [n = 85, 87, 79, and 77 animals, respectively; N = 5 biologically independent experiments; statistical analysis is by one-way ANOVA, followed by Tukey’s post-test (****p ≤ 0.0001)]. All bar graphs represent mean + SEM. Arrowhead on y-axis of all liquid thrashing bar graphs denotes non-Tg animals average ~70 thrashes/min under standard laboratory conditions. Source data are available as Supplementary Data.

The C. elegans gene F41E7.6 is an ortholog of human CROT and is predicted to encode a carnitine O-octanoyltransferase (CROT) in the carnitine acyltransferase family of enzymes. CROT catalyzes the transfer of medium- and long-chain fatty acyl groups from coenzyme A (CoA) to L-carnitine to facilitate transport of these molecules out of the peroxisome and into the cytosol and mitochondria for further fatty acid metabolism⁷⁷. When compared to non-Tg animals, F41E7.6 (−/−) animals [F41E7.6 (tm5587)] had mild locomotion defects (Supplementary Fig. 2). Loss of function of F41E7.6 in the Tau (high) background did not alter tau-induced locomotion abnormalities when compared to Tau (high) animals alone (Fig. 6b). However, when F41E7.6 (−/−) was crossed to Tau (high); xbp-1s Tg animals, xbp-1s overexpression failed to suppress tau-induced behavioral defects, indicating that F41E7.6 was also necessary for this mechanism (Fig. 6b).

C01B4.6 is a C. elegans ortholog of the human gene GALM, which encodes a galactose mutarotase (GALM), also known as aldose 1-epimerase. This enzyme converts β- to α-D-galactose (and, to a much lesser extent, glucose) for downstream metabolism⁷⁸. When compared to non-Tg animals, C01B4.6 (−/−) animals [C01B4.6 (tm6913)] had mild locomotion defects (Supplementary Fig. 2). Loss of function of C01B4.6 in Tau (high) animals had no effect on tau-induced behavioral deficits compared to Tau (high) animals alone (Fig. 6c). However, crossing C01B4.6 (−/−) with Tau (high); xbp-1s Tg animals resulted in loss of xbp-1s-mediated suppression of tau-induced locomotion impairment (Fig. 6c). Y19D10A.16 is another C. elegans ortholog of the human gene GALM. When compared to non-Tg animals, Y19D10A.16 (−/−) animals [Y19D10A.16 (bk3109)] had mild locomotion defects (Supplementary Fig. 2). Deletion of this gene in Tau (high) animals resulted in behavioral deficits similar to Tau (high) alone (Fig. 6d). However, loss of Y19D10A.16 reversed the behavioral rescue seen in Tau (high); xbp-1s Tg animals (Fig. 6d), indicating both GALM orthologs (C01B4.6, Y19D10A.16) are also necessary for xbp-1s to suppress tau-induced motor impairment.

The C. elegans gene eol-1 is orthologous to the human gene DXO, which encodes a decapping exoribonuclease (DXO) with pyrophosphohydrolase, decapping, and 5′-3′ exoribonuclease activity⁷⁹. DXO contributes to RNA capping quality control by removing incomplete 5’ N7-methylguanosine (m7G) or non-canonical nicotinamide adenine dinucleotide (NAD), flavin adenine dinucleotide (FAD), and dephospho-CoA caps from RNA^80,81. To investigate whether eol-1 is required for xbp-1s-mediated tauopathy suppression in C. elegans, we used an eol-1 loss-of-function mutant strain [eol-1 (gk534833); referred to as eol-1 (−/−)] crossed to Tau (high); xbp-1s Tg animals. When compared to non-Tg animals, eol-1 (−/−) animals had mild locomotion defects (Supplementary Fig. 2). Loss of function of eol-1 in Tau (high) animals had no effect on tau-induced locomotion impairment when compared to Tau (high) animals alone (Fig. 6e). However, eol-1 loss of function blocked the ability of xbp-1s Tg to suppress tau-induced behavioral defects in Tau (high); xbp-1s Tg; eol-1 (−/−) animals when compared to Tau (high); xbp-1s Tg animals (Fig. 6e), which indicates eol-1 is also required for xbp-1s- mediated suppression of tauopathy.

The C. elegans gene mct-2 is orthologous to human SLC16A4 in the monocarboxylate transporter family, also known as the solute carrier 16 (SLC16) family^82–84. Its protein is predicted to transport monocarboxylic acids such as pyruvate, lactate, and ketone bodies across the plasma membrane. When compared to non-Tg animals, mct-2 loss of function [mct-2 (bk3105); referred to as mct-2 (−/−)] modestly enhances locomotion (Supplementary Fig. 2). While deletion of mct-2 does not affect tau-induced motility defects in Tau (high) animals, it blocks suppression of these defects in Tau (high); xbp-1s Tg animals (Fig. 6f). Therefore, xbp-1s also requires functional mct-2 to suppress tauopathy phenotype.

Taken together, many C. elegans genes with human homologs (Table 1), which function in disparate molecular pathways, are required for xbp-1s overexpression to suppress tau-induced behavioral deficits in C. elegans. In addition to the five genes identified with a previously known association with ATF6 (csp-1, dnj-28, hsp-4, ckb-2, and lipl-3; Table 2), each of the six XBP-1s target genes without previous association with ATF6 (erp-44.3, F41E7.6, C01B4.6, Y19D10A.16, eol-1, and mct-2; Table 1) must be intact for XBP-1s-mediated suppression of tauopathy in C. elegans. These results indicate ATF-6 association may not be necessary for determining which downstream targets of XBP-1s are required to ameliorate disease phenotype. Furthermore, these results support the robustness of the XBP-1s transcriptional analysis in C. elegans and provide information on potential mediators of tauopathy.

XBP1s induction upregulates tauopathy-related target genes, but causes lethality in mice

To assess the translational relevance of UPR^ER hyperactivation in the mammalian brain, we generated a mouse model conditionally overexpressing mouse XBP1s specifically in neurons. To produce these mice, we employed the tetracycline reverse transcriptional activator (rtTA)-driven conditional XBP1s transgene previously generated⁸⁵ as a responder transgene and crossed them to the previously generated neurofilament heavy chain (NEFH) promoter driver transgene^86,87 to yield double transgenic neuron-inducible XBP1s (niXBP1s) mice (Fig. 7a). Double transgenic mice appear viable and healthy when transgene expression is suppressed by tetracycline. Young adult niXBP1s mice exhibit abundant XBP1s protein readily detectable in hippocampal neurons by 10 days after transgene induction (Fig. 7b). We evaluated a subset of the mouse homologs of candidate C. elegans xbp-1s target genes identified above and observed HSPA5/BiP, the mouse homolog of hsp-4, becomes dramatically upregulated in hippocampal neurons in niXBP1s mice, confirming conservation of hsp-4/BiP as an XBP-1s target gene across phyla (Fig. 7c). Similarly, DNAJC3, the mouse homolog of C. elegans xbp-1s target gene dnj-28, becomes induced in the hippocampus of niXBP1s mice (Fig. 7d). While initially healthy, niXBP1s mice exhibit a sudden death phenotype between 2–3 weeks post-induction, precluding the long-term evaluation of niXBP1s impacts on aging-related tauopathy phenotypes. We suspect this early death phenotype is driven by sustained UPR^ER activation and subsequent ER stress-induced apoptosis through the CHOP pathway^88,89. We attribute the long-term tolerability of XBP-1s expression in C. elegans neurons to the absence of a homolog of CHOP in the C. elegans genome⁶⁰.

Fig. 7 — a Schematic diagram of Dox-regulatable pan-neuronal expression of XBP1s in bigenic mice under the control of the *NEFH* promoter. b XBP1s induction is detectable by immunohistochemistry in niXBP1s mice off Dox. Representative hippocampal brain sections from 4-month-old non-Tg and niXBP1s mice stained with XBP1s antibody are shown. c BiP, the mouse homolog of *C. elegans hsp-4*, is upregulated in niXBP1s mice off Dox. Representative hippocampal brain sections from 4-month-old non-Tg and niXBP1s mice stained with BiP/HSPA5 antibody are shown. d DNAJC3, the mouse homolog of *C. elegans dnj-28*, is upregulated in niXBP1s mice off Dox. Representative hippocampal brain sections from 4-month-old non-Tg and niXBP1s mice stained with DNAJC3 antibody are shown. Scale bars: 500 µm. Schematic diagram was created with BioRender.

Discussion

Aging-related declines in cellular function and neurodegenerative diseases are closely associated with overall cellular proteostasis, including the UPR^ER. Much work has been conducted to understand the role of the UPR^ER in the context of health and disease over the lifespan. However, contradictory results from studies in neurodegenerative tauopathies warrants a more direct approach to dissect the underlying mechanisms of the UPR^ER in tauopathy. Because the three stress transducers of the UPR^ER each control transcriptional regulation via XBP1s, ATF6n, and ATF4 transcription factors, understanding the role of downstream upregulated target genes is important for identifying unique regulators of tauopathy. Here, we followed up on our previous study³⁰ by exploring the role of UPR^ER transcriptional regulation via XBP-1s in human wild type tau transgenic C. elegans.

Constitutive activation of the master UPR^ER transcription factor XBP-1s suppresses tauopathy phenotypes in C. elegans, which relies on functional ATF-6³⁰. Because XBP-1s is essential for UPR^ER transcriptional remodeling, we conducted whole organism transcriptomic analysis at the L2 developmental stage to enrich for neuronal transcripts upregulated by XBP-1s in Tau (high) C. elegans. We narrowed our search to find candidate XBP-1s target genes in three sequential steps: identifying significant genes upregulated in xbp-1s Tg animals compared to non-Tg animals (116 genes; Supplementary Table 1), identifying significant genes with human homologs (11 genes; Table 1), and identifying significant genes with human homologs and a known association with ATF6 (5 genes; Table 2). From this analysis, we nominated multiple genetic modifiers of tauopathy associated with XBP-1s/ATF6 in C. elegans, including csp-1, dnj-28, hsp-4, ckb-2, and lipl-3.

Another study conducted intestine-specific RNA sequencing analysis in C. elegans to identify genes upregulated in the intestine when XBP-1s is overexpressed in neurons through non-cell autonomous UPR^ER signaling⁹⁰. Neuronal XBP-1s significantly increased the expression of genes involved in lysosome function, and intestinal lysosome function was necessary for enhanced lifespan and proteostasis. Compared to genes in Table 1, dnj-28, erp-44.3, hsp-4, and ckb-2 were also upregulated in this RNA sequencing dataset. Consistent with our results regarding the lipase family member lipl-3 gene, lipl-1, lipl-2, and lipl-5 were also significantly upregulated in the intestine in response to neuronal xbp-1s Tg overexpression. However, csp-1, F41E7.6, C01B4.6, Y19D10A.16, mct-2, and eol-1 were not identified as significantly upregulated or downregulated. These differences indicate a potential divergence of neuronal XBP-1s in tauopathy suppression versus lifespan extension, which warrants further investigation.

To genetically dissect the contributions of each XBP-1s/ATF6 candidate gene, we systematically eliminated the function of csp-1, dnj-28, hsp-4, ckb-2, and lipl-3 in Tau (high); xbp-1s Tg C. elegans. We supported the validity of the transcriptomic analysis by showing putative loss of function of five candidate genes abolished xbp-1s-mediated tauopathy suppression in C. elegans (Figs. 2–5). To fully understand the specificity of these nominated genes in C. elegans tauopathy, we tested whether another member of the same functional gene classification affected Tau (high); xbp-1s Tg animal phenotypes. In contrast to hsp-4 loss of function (Fig. 4a and Supplementary Fig. 5), hsp-3 (−/−) did not affect xbp-1s-mediated behavioral tauopathy suppression (Fig. 4d). Interestingly, ced-3 (Fig. 2d) and dnj-27 (Fig. 3d) are also required for tauopathy suppression via xbp-1s, indicating a potential broader role of caspase and DNAJ domain genes in xbp-1s-mediated tauopathy suppression.

Additionally, we tested whether the nominated XBP-1s target genes could modulate tauopathy in C. elegans in the absence of xbp-1s-mediated UPR^ER induction. We show hsp-4 loss of function enhanced behavioral tauopathy phenotypes without affecting total tau protein (Fig. 4e–g), while dnj-28 loss of function does not regulate tauopathy in Tau (low) animals (Fig. 3e). We also generated neuronal hsp-4 overexpression transgenic C. elegans models and crossed these with Tau (high) animals. We show low expression of hsp-4 modestly suppressed tauopathy behavioral phenotypes, while hsp-4 high expression had no effect or modestly enhanced behavioral phenotypes (Fig. 4h, i). Interestingly, hsp-4 low overexpression did not affect total tau protein, while hsp-4 high overexpression increased total tau protein in the Tau (high) background (Fig. 4j, k). Given the intricate nature of XBP-1s-mediated transcriptional remodeling, future work should explore loss of function and gain of function of all candidate target genes in tauopathy suppression.

Finally, we also investigated putative loss of function of XBP-1s candidate genes with human homologs, but without previous association with ATF6: erp-44.3, F41E7.6 (CROT ortholog), C01B4.6 (GALM ortholog), Y19D10A.16 (GALM ortholog), eol-1, and mct-2. Surprisingly, xbp-1s-mediated suppression of tauopathy also requires each of these genes (Fig. 6). Given their varied subcellular localization and functions, xbp-1s activates a diverse and non-redundant network of genes required for tauopathy suppression in C. elegans (Table 1 and Fig. 8).

Fig. 8 — Schematic diagram showing the 13 genes comprising four components of the *xbp-1s* transcriptional network and subcellular localization required for tauopathy suppression in *C. elegans*: protein folding (*dnj-27*, *dnj-28*, *eol-1*, *erp-44.3*, and *hsp-4*), lipid metabolism (*ckb-2*, *F41E7.6*, *lipl-3*, and *mct-2*), carbohydrate metabolism (*C01B4.6* and *Y19D10A.16*), and apoptosis (*ced-3* and *csp-1*). Schematic diagram was created with BioRender.

The first component of the proposed xbp-1s transcriptional network involves protein folding target genes. The most apparent network members participate in protein processing capacity in the UPR^ER and include hsp-4, dnj-28, dnj-27, and erp-44.3. The C. elegans homolog of BiP, HSP-4, senses misfolded proteins and initiates the UPR^ER. Consequently, hsp-4 low overexpression improves tau-induced locomotor defects in the Tau (high) C. elegans model (Fig. 4h, i), presumably by acting downstream of UPR^ER activation. DNAJC3, the mammalian homolog of DNJ-288, can recruit BiP to the ER membrane and help degrade misfolded proteins. It also increases ER protein folding capacity by inhibiting PERK, which downregulates translation⁶⁵. DNAJC3 knockout in mice results in an inability to cope with ER stress, and in humans causes multisystemic neurodegeneration and diabetes mellitus⁹¹, suggesting an integral role in ER and cellular processing of proteins. Since ERP44 acts as a sentry for mislocalized enzymes and disulfide-linked oligomeric proteins between the ER and Golgi, we would expect its upregulation to enhance cellular capacity for protein quality control. Altogether, these three genes work directly to improve proteostasis in the cell.

As a decapping exoribonuclease, eol-1 appears to represent a distinct pathway downstream of xbp-1s. However, mRNA cap-independent translation has been demonstrated to be a feature of UPR^ER-mediated gene expression changes in UPR^ER-regulated fatty acid biosynthesis⁹². Further, regulation of C. elegans hsp-4 and cell death genes has been demonstrated to occur through a cap-independent mechanism⁹³. Thus, eol-1 could indirectly support the functions of chaperones by enhancing their translation in the face of ER stress. The action of eol-1 may also ameliorate the UPR^ER by degrading mRNAs encoding secretory proteins targeted to the ER, thereby reducing protein load while enriching for ER resident chaperones to enhance the UPR^ER capacity to clear misfolded proteins.

We propose a second component of the xbp-1s transcriptional network for lipid metabolism involving lipl-3, mct-2, F41E7.6 (CROT ortholog), and ckb-2. These genes may be involved in xbp-1s-induced lipid remodeling, which increases longevity and protects against proteotoxic stress and accumulation of misfolded protein species in C. elegans^94,95. This lipid gene expression remodeling may also reflect xbp-1s-induced ER expansion⁷⁴, which would involve ckb-2, or the UPR^ER transcriptional response to lipid bilayer stress⁹⁶.

The third component of the proposed xbp-1s transcriptional network involves carbohydrate metabolism. Our transcriptomic dataset yielded two C. elegans homologs of GALM (C01B4.6, Y19D10A.16), providing convincing evidence this gene is indeed upregulated in response to xbp-1s overexpression. While involvement of GALM in the UPR^ER remains unexplored, this enzyme is upstream of uridine diphosphate (UDP)-galactose 4-epimerase in the Leloir pathway, which converts galactose to glucose. In mammals, XBP1s regulates UDP-galactose 4-epimerase expression in the liver for protein glycosylation under fed conditions⁸⁵. Therefore, an increased expression of GALM may reflect an upregulation of the Leloir pathway in xbp-1s Tg C. elegans through a transcriptional program distinct from pathological UPR^ER activation and represent signal from non-neuronal cells as a result of whole-animal RNA sequencing⁹⁷ or a C. elegans-specific pathway. Indeed, non-cell autonomous signaling by XBP-1s between neurons and the intestine in C. elegans promotes stress resistance and longevity^32,98, so it seems unsurprising to detect the downstream transcriptional effects of neuronal XBP-1s overexpression in peripheral tissues.

Lastly, we propose a fourth component of the xbp-1s transcriptional network for apoptosis involving csp-1 and ced-3, which are homologous to the human CASP family of genes. C. elegans possess four caspases: CED-3, CSP-1, CSP-2, and CSP-3. Of these, CED-3 is an essential C. elegans caspase required for cell death⁹⁹. However, further work has shown CSP-1B can cleave the CED-3 proprotein in vitro, suggesting C. elegans apoptosis may involve a proteolytic cascade¹⁰⁰, and CSP-1B can kill cells mostly independent of CED-3⁶⁴. In mammals, CASP3, 6, and 7 are considered the executioners in apoptosis¹⁰¹. Chronic ER stress upregulates CASP3¹⁰² and induces apoptosis¹⁰³. CASP6 and CASP6-cleaved tau are found in tangles and neurites of AD frontal and temporal cortex¹⁰⁴, while immunodetection of CASP3 is limited to hippocampal neurons undergoing granulovacuolar degeneration¹⁰⁵. Both CASP3 and 6 colocalize with hyperphosphorylated tau in AD brainstem¹⁰⁶.

The absolute requirement for each one of the major XBP-1s target genes for tauopathy suppression in C. elegans is truly surprising. While the necessity of inducible BiP expression as a component of canonical UPR^ER function was fully anticipated, the same level of involvement for each of the other XBP-1s target genes (Table 1 and Figs. 2–6) was not an expected outcome. We do not currently have a conceptual framework for the UPR^ER transcriptional network that adequately explains these observations. On the surface, we take these observations as support for a hitherto undescribed molecular feedback mechanism for the UPR^ER whereby XBP-1s target genes reinforce UPR^ER-mediated tauopathy rescue through a transcriptional program. However, the molecular mechanism for such a diverse set of proteins to all mediate UPR^ER transcriptional activity does not seem readily apparent. Neither does downstream involvement of all Table 1 target genes seem likely to be directly linked to tau proteostasis, as we show for dnj-28 loss of function in the Tau (low) background (Fig. 3e). Thus, we plan to continue the molecular investigation of this phenomena through a classical forward genetic approach in C. elegans beyond the scope of this transcriptomic study.

The C. elegans model system provides many advantages for dissecting the underlying mechanisms of pathology in human neurodegenerative diseases that are not easily achieved in intact mammalian model systems. However, to understand the translational capability of UPR^ER activation in mammals, we generated niXBP1s mice (Fig. 7a). We found robust expression of XBP1s and two target gene proteins (BiP and DNAJC3) in hippocampal neurons of niXBP1s mice (Fig. 7b–d). Another group also recently generated a conditional and neuron-inducible XBP1s transgenic mouse model using a different driver transgene promoter, calcium/calmodulin dependent protein kinase II alpha (CAMK2A)¹⁰⁷. Consistent with the sudden death phenotype observed in niXBP1s mice, these mice exhibited progressively worsening seizures followed by death approximately 2 weeks after induction of Xbp1s overexpression¹⁰⁷.

In contrast to these unexpected sudden death phenotypes resulting from sustained overexpression of XBP1s in neurons, prior XBP1s overexpression studies in mice showed overall beneficial effects on brain function. One group generated a transgenic mouse model which constitutively expresses moderate levels of XBP1s under the control of the prion promoter¹⁰⁸. These mice exhibited improved basal learning capacity associated with improved long-term potentiation (LTP) and synaptic transmission in the hippocampus¹⁰⁸. In a follow-up study, this group also found XBP1s overexpression reduced Aβ deposition and preserved synaptic and cognitive function in the AD mouse model expressing five familial AD mutations (5xFAD)¹⁰⁹. Additionally, local delivery of XBP1s via adeno-associated vectors into the hippocampus of 5xFAD mice after Aβ has begun accumulating also restored cognitive function and synaptic plasticity¹⁰⁹. Another group used Xbp1s-expressing viral vectors delivered into the hippocampus of triple transgenic AD model mice (3xTg-AD; a less aggressive AD model compared to 5xFAD)¹¹⁰. In this AD mouse model, XBP1s overexpression prevented the loss of dendritic spines and improved neuronal plasticity¹¹⁰. Differences in the degree of expression, sustained versus transient overexpression, or cell type specificity of expression related to gene promoters used could explain the contrasting phenotypes observed among these different XBP1s mouse models. Regardless, we believe the absence of a CHOP encoding gene in the C. elegans genome underlies the lack of toxic phenotype by prolonged xbp-1s activation in C. elegans neurons and the presence of CHOP drives the dose-dependent toxicity in mice discussed above.

Recently, it was shown ER chaperones act downstream of the transcription factor nuclear receptor subfamily 4, group A, member 1 (Nr4a1) to effect changes in synaptic plasticity required for long-term memory in mice¹¹¹. Blocking Nr4a1 transcriptional activity resulted in downregulation of pathways related to ER protein processing, chaperone binding, misfolded protein binding, and PDI activity. These genes include Xbp1, Hspa5, Dnajc3, and members of the Pdi family, which overlap with C. elegans homologs in our transcriptomic dataset and are required for xbp-1s-mediated suppression of tauopathy. Additionally, overexpression of Nr4a1 or Hspa5 improved long-term memory in a tau-based mouse model of AD. Together with our findings, these results begin to describe a mechanism whereby upregulation of xbp-1s target genes can rescue neurological deficits.

Taken altogether, XBP-1s activates a network of genes involved in protein processing, lipid remodeling, carbohydrate metabolism, and apoptosis, all of which are required for xbp-1s-mediated suppression of tauopathy in C. elegans (Fig. 8). These data support a model where multiple xbp-1s target genes act in a UPR^ER transcriptional target network to ameliorate tauopathy. Among this network of target genes, hsp-4/BiP exhibits a prominent impact independent of other XBP-1s target genes that appears conserved across animal phyla as diverse as Nematoda and Mammalia. Given the intricate nature of XBP-1s-mediated transcriptional remodeling, future work should explore the functional interrelationships between UPR^ER-regulated genes to mediate protection from pathological tau in disease. This will provide a deeper mechanistic understanding of XBP-1s-mediated neuroprotection in C. elegans tauopathy models and direct further studies in mammalian model systems, with the goal of modulating tauopathy in humans.

Methods

Plasmids

To generate transgenes expressing HSP-4 driven by a pan-neuronal snb-1 promoter, a double stranded gene fragment of full-length C. elegans hsp-4 was designed (gBlock Gene Fragment; Integrated DNA Technologies, Inc., Coralville, IA, USA). The hsp-4 gBlock Gene Fragment served as a fragment for Gibson assembly¹¹² using the snb-1p vector linearized with SacI and KpnI restriction enzymes.

C. elegans strains and transgenics

C. elegans strains used are listed in Supplementary Table 3. All strains were maintained at 20 °C on standard nematode growth media (NGM) plates containing OP50 Escherichia coli (E. coli)¹¹³. To enhance animal yield for biochemical and molecular analysis, C. elegans were grown on NGM plates containing five times more peptone (NGM 5X PEP plates) prior to collection for protein and RNA extraction using established methods^31,114,115. The C. elegans husbandry and experimentation was conducted in accordance with all relevant ethical and safety regulations for animal testing and research.

The hsp-4 (high) and hsp-4 (low) transgenic C. elegans strains were engineered by microinjection using a Nikon Eclipse TE300 microscope (Nikon Instruments, Inc., Melville, NY, USA) and Eppendorf FemtoJet® electronic microinjector setup (Eppendorf, Hamburg, Germany) into N2 (non-Tg) C. elegans at a concentration of either 100 ng/µl snb-1p::hsp-4; 20 ng/μl myo-3p::mCherry; 50 ng/μl pUC19 carrier DNA or 50 ng/μl snb-1p::hsp-4; 30 ng/μl myo-3p::mCherry; 70 ng/μl pUC19 carrier DNA to produce worms carrying extrachromosomal arrays. Day one adult animals from these generated lines were irradiated for 48 s with a UV lightsource in a Stratalinker© UV Crosslinker 1800 (Stratagene, San Diego, CA, USA), after two sequential autocrosslink warm-ups to integrate the extrachromosomal array into the genome. Irradiated worms were separated onto 150 mm NGM 5X PEP plates and hard starved for several weeks to enhance detection of transgene genomic integration events^36,58. Starved populations were washed onto fresh plates and a portion of live, fluorescently marked animals were recovered to individual plates and screened for integration. Successfully integrated lines were screened by isolating individual worms with 100% transmission of the myo-3p::mCherry marker and outcrossed with N2 males at least two times. Custom loss of function alleles in Supplementary Table 3 were generated using CRISPR-Cas9 genome editing technology by introducing purified active recombinant Cas9 protein and synthetic CRISPR guide RNAs (gRNAs)^49,116.

C. elegans behavioral analysis

Manual swimming assay

C. elegans swimming behavior was assessed using established methods³⁶. C. elegans were developmentally synchronized using a timed egg-lay and grown until all reached day one of adulthood at 20 °C. Individual developmentally synchronized animals were placed in a 10 µl M9 buffer droplet on a 10 well Teflon-printed glass slide and allowed to acclimate to a liquid environment for 10 s. One thrash was defined as a bend across the midline or two consecutive bends from the midline toward the same side. The number of thrashes were counted during a one minute period and averaged for each strain. At least three biological replicate assay sessions with at least 10 animals per assay session were analyzed for statistical significance. Behavioral analysis was conducted blinded to genotype to avoid biases. However, when the strains to be assayed were visibly distinguishable, blinding was not feasible. For comparison of two groups, an unpaired t-test, two-tailed, was used. For comparisons of three or more groups, a one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison post-test, was used.

Automated swimming assay

Supplementary data was acquired using an automated platform for measuring C. elegans swimming behavior⁴⁷. C. elegans were developmentally synchronized using a timed egg-lay and grown until all reached day one of adulthood at 20 °C. Animals were moved to the assay room and allowed to acclimate for at least 30 min. NGM plates were flooded with 1 ml M9 buffer and transferred to 35 mm NGM video plates lacking OP50 E. coli. Animals acclimated to M9 buffer for 10 s prior to a one minute video recording. Videos were acquired using the WormLab platform (MBF Bioscience, Williston, VT, USA). After videos were taken, worm movement behavior was analyzed using the WormTracker software (MBF BioScience, version 2020.1.1). The frequency of body bends, defined as turns by the software, was quantified as follows. A turn was defined as a change in the body angle defined by the quarter points and midpoint of the animal that was at least 20 degrees positive or negative from a straight line. Animals that were tracked for less than 30 s were omitted from analysis. The total number of body bends was divided by the track duration to give the frequency of body bends per second. This was multiplied by 60 s to give the frequency of body bends per minute. At least three biological replicate assay sessions with roughly 15–60 animals per assay session were analyzed for statistical significance. For comparison of two groups, an unpaired t-test, two-tailed, was used. For comparisons of three or more groups, a one-way ANOVA, followed by Tukey’s multiple comparison post test, was used.

Radial locomotion assay

C. elegans locomotion was assessed using published methods¹¹⁷. C. elegans were developmentally synchronized using a timed egg-lay and grown until all reached day one of adulthood at 20 °C. Developmentally synchronized animals were placed at the center of a 100 mm NGM 5X PEP plate. Animals were allowed to move freely for 1 h, and the radial distance traveled from the start point was recorded as radial dispersion in mm. At least two biological replicate assay sessions with at least 10 animals per assay session were analyzed for statistical significance. For comparison of three groups, a one-way ANOVA, followed by Tukey’s multiple comparison post-test, was used.

C. elegans protein extraction

Protein was extracted from C. elegans using a rigorous lysis approach³⁶. C. elegans were synchronized using hypochlorite treatment and grown until adulthood at 20 °C on NGM 5X PEP plates. Synchronized adult C. elegans were washed off NGM 5X PEP plates in M9 buffer, washed an additional four times in M9 buffer to remove excess OP50 E. coli, pelleted by a one minute centrifugation at 800 × g, snap frozen in liquid nitrogen, and stored at −70 °C until protein extraction. A total of 2 µl of high-salt reassembly (RAB) buffer (0.1 M 2-(N-morpholino)ethanesulfonic acid, 1 mM ethylene glycol bis-2-aminoethyl ether-N,N’,N”,n’-tetraacetic acid, 0.5 mM MgSO₄, 0.75 M NaCl, 0.02 M NaF, pH 7.0) containing phenylmethylsulfonyl fluoride and protease inhibitors was added per milligram of packed worm pellet wet weight and homogenized via sonication four times at 70% power for eight seconds. This total soluble worm lysate was reserved for subsequent immunoblotting.

Protein immunoblotting

C. elegans protein immunoblotting was performed using Criterion apparatus (Bio-Rad) as recommended by the manufacturer (Bio-Rad Laboratories, Hercules, CA, USA)³⁶. C. elegans protein preparations were diluted 1:5 (w/v) with sample buffer (0.046 M Tris, 0.005 M ethylenediaminetetraacetate, 0.2 M dithiothreitol, 50% sucrose, 5% sodium dodecyl sulfate, 0.05% bromophenol blue), sonicated for 15 s three times at 70% microtip power, boiled for 10 min, and centrifuged at 16,100 × g for 1–2 min. A total of 10 µl of lysate preparation (representing ~2 µg of protein) were loaded and resolved on precast 4 to 15% gradient SDS-PAGE gel and transferred to PVDF membrane as recommended by the manufacturer (Bio-Rad Laboratories, Hercules, CA, USA). PVDF membranes were blocked in 5% milk in PBS for 1 h before overnight incubation with primary antibody at 4 °C. The next day, PVDF membranes were washed in PBS with 0.1% Tween, incubated at room temperature with HRP-coupled secondary antibody for 2 h, and washed in PBS with 0.1% Tween before visualization. The dilutions and pertinent details for all primary and secondary antibodies used are listed in Supplementary Table 4. Enhanced chemiluminescence substrate (Bio-Rad Laboratories, Hercules, CA, USA) was added to the PVDF membrane, and chemiluminescence signals were detected with ChemiDocIt®² 510 Imager (UVP LLC, Upland, CA, USA) or LiCor Odyssey Fc 2800 (LI-COR Biosciences, Lincoln, NE, USA). Relative intensity of chemiluminescence signals was measured with ImageJ Java¹¹⁸. At least three biological assay replicates were analyzed for statistical significance. For comparison of two groups, an unpaired t-test, two-tailed, was used. For comparison of three or more groups, a one-way ANOVA, followed by Tukey’s multiple comparison post-test, was used.

C. elegans RNA purification

To achieve a tightly synchronized C. elegans population, two rounds of synchronization were completed. First, mixed population C. elegans were synchronized using hypochlorite treatment and grown until adulthood at 20 °C on NGM 5X PEP plates. From this adult population, C. elegans were synchronized using hypochlorite treatment and grown until L2 larval stage at 20 °C on NGM 5X PEP plates. Synchronized L2 C. elegans were washed off NGM 5X PEP plates in M9 buffer, washed an additional four times in M9 buffer to remove excess OP50 E. coli, pelleted by a one minute centrifugation at 800 × g, snap frozen in liquid nitrogen, and stored at −70 °C until RNA extraction. RNA was purified from snap-frozen packed C. elegans pellets using TRIzol Reagent as directed by the manufacturer’s instructions⁵⁴. RNA was resuspended in 50 ul sterile water. RNA concentration and purity were assessed using a NanoPhotometer® NP80 spectrophotometer (Implen GmbH, Munich, Germany). RNA integrity was assessed by 1% TBE agarose gel electrophoresis.

Library construction and transcriptomic analysis

C. elegans cDNA libraries for next generation sequencing were prepared from purified total RNA using the TruSeq reagents (Illumina, Inc., San Diego, CA, USA) at the Northwest Genomics Center within the University of Washington, Department of Genome Sciences. Libraries were sequenced using Illumina NovaSeq technology (Illumina, Inc., San Diego, CA, USA) with at least 25 million reads per sample. Three biological replicates with two technical replicates for each sample were analyzed. Transcriptomic analysis was completed using the Lasergene bioinformatics software version 17 (DNASTAR, Inc., Madison, WI, USA). The sequencing data was aligned to the C. elegans genome (release WB235) using Lasergene SeqMan NGen software (DNASTAR, Inc., Madison, WI, USA). Subsequent differential gene expression analysis was completed using Lasergene ArrayStar software (DNASTAR, Inc., Madison, WI, USA). Comparison of non-Tg and xbp-1s Tg animals yielded 2697 upregulated genes with two-fold or greater differential gene expression levels in xbp-1s Tg versus non-Tg animals. Among these genes, 560 genes exhibited significant gene expression changes in all four groups, analyzed by a two-way ANOVA, followed by a Benjamini-Hochberg post-test, with 131 of these genes exhibiting an F-value greater than 10. Filtering this gene set for genes with xbp-1s levels greater than 0.1 Reads Per Kilobase Million (RPKM) yielded a total of 116 significant genes in this analysis (Supplementary Table 1). The group of xbp-1s target genes was analyzed using the Basic Local Alignment Search Tool (BLAST)¹¹⁹ to identify human orthologs (BLAST expected values <1 × e⁶), yielding 11 significant genes (Table 1) and 5 significant genes with literature evidence of an association with ATF6 (Table 2). Gene Ontology (GO) term enrichment analysis was conducted using the Database for Annotation and Integrated Discovery (DAVID)¹²⁰. Graphics representing analysis were generated using SRplot webtools¹²¹.

Work with mice

Work with mice was reviewed and approved by the VA Puget Sound Health Care System Institutional Animal Care and Use Committee (IACUC) and conducted in an American Association for Accreditation of Laboratory Animal Care (AAALAC)-accredited animal research facility. We have complied with all relevant ethical regulations for animal use. C57BL/6J (non-Tg) was used as the control strain of mice, both sexes were used for study, and all strains used were maintained in a congenic state on this background. niXBP-1s mice were made by cross breeding two previously generated strains: the NEFH driver strain^86,87 with the XBP-1s responder transgene⁸⁵. For tissue harvest, mice were anesthetized and fixed by transcardial perfusion with 4% paraformaldehyde. Brains were removed and paraffin embedded for sectioning. Coronal sections (9 microns) were prepared and stored at 4 °C until use.

Immunohistochemical evaluation

Animal brain sections were deparaffinized, rehydrated through alcohols, and processed through antigen retrieval steps consisting of heat pretreatment in citrate buffer by either microwave or autoclave per antibody-specific protocols. Sections were treated for endogenous peroxidases with 3% hydrogen peroxide for 30 min at room temperature, blocked in 5% non-fat milk in PBS for 1 h at room temperature, and incubated with primary antibodies overnight at 4 °C. Biotinylated secondary goat anti-mouse or goat anti-rabbit antibody was applied for 45 min at room temperature. The dilutions and pertinent details for all primary and secondary antibodies used are listed in Supplementary Table 4. Finally, sections were incubated in an avidin-biotin complex with streptavidin-HRP (Vector’s Vectastain Elite ABC-HRP kit, Burlingame, CA, USA) for 1 h at room temperature and the reaction product was visualized with 0.05% diaminobenzidine (DAB)/0.01% hydrogen peroxide in PBS. Negative controls consisted of full protocol except primary antibody. Digital images were obtained using a Leica DM6 microscope with a DFC 7000 digital camera (Leica Microsystems, Wetzlar, Germany) and imported into Adobe Photoshop (Adobe Inc, San Jose, CA, USA).

Statistics and reproducibility

Statistical significance for all phenotype assays was determined using GraphPad Prism version 10.1.0 statistical software (GraphPad Software, Inc., Boston, MA, USA). Statistical significance is demarcated in figures as ns: p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. The details about experimental design and statistics used in different data analyses performed in this study are given in the respective sections of methods and figure legends.

Schematic illustrations

Schematic illustrations in Figs. 1a, b, 7a and 8 were created using an academic license with the online application BioRender (Toronto, ON, Canada).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary Information^{(1.3MB, pdf)}

42003_2024_6570_MOESM2_ESM.pdf^{(26.4KB, pdf)}

Description of Additional Supplementary Files

Supplementary Data 1^{(1.2MB, xlsx)}

Reporting Summary^{(2.1MB, pdf)}

Acknowledgements

We thank Jade Stair, Nzinga Hendricks, Lisa Chiang, and Brandon Henderson for essential technical assistance, including pouring plates and making media/reagents. We thank Rebecca Kow, Heather Currey, Vaishnavi Jadhav, and Elaine Loomis for sharing their expertise. We thank Jeremy Baker and Richard Gardner for outstanding mentorship, advice, and personal support of this work (in memoriam). We thank Shohei Mitani, the Japan National Bioresource Project (NBRP), the C. elegans Genetics Center [CGC; funded by National Institute of Health’s Office of Research Infrastructure Programs (P40 OD010440)], and the Reverse Genetics Core Facility at the University of British Columbia (part of the international C. elegans Gene Knockout Consortium) for providing C. elegans loss of function strains. We thank Rebecca Taylor and Andrew Dillin for providing xbp-1s C. elegans strains and advice about UPR activation in C. elegans. We thank WormBase for essential C. elegans model organism information. We thank Phillip Scherer for providing the iXBP1s mouse responder transgene strain. We thank the Developmental Studies Hybridoma Bank (NICHD) for the β-tubulin primary antibody E7. We thank Peter Davies (Feinstein Institute of Medical Research) for CP13 and PHF-1 tau phosphorylation epitopes primary antibodies. This work was supported by the Department of Veterans Affairs and National Institutes of Health grants IK6BX006467 (B.C.K.), R01AG066211 (B.C.K.), R01NS064131 (B.C.K.), R01AG066729 (N.F.L.), I01BX004044 to (N.F.L.), and T32-AG052354 (S.M.W.).

Author contributions

S.M.W. performed experiments, analyzed data, and wrote the manuscript. M.H. performed experiments, analyzed data, and wrote the manuscript. A.D.S. performed experiments. T.A.V. performed experiments. P.J.M. performed experiments and analyzed data. J.M.W. performed experiments. N.F.L. wrote the manuscript. B.C.K. conceived and oversaw the study, analyzed data, and wrote the manuscript. All authors contributed to the final version of the manuscript.

Peer review

Peer review information

Communications Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editors: Asuka Takeishi and Manuel Breuer.

Data availability

The data analyzed for this study are published in this manuscript and associated Supplementary Information. The source data underlying the graphs in Figs. 2–6 and Supplementary Figs. 1–6, 8, 9 are available as a spreadsheet file called: Supplementary Data 1. The source data for uncropped full immunoblot images underlying Figs. 2b, e, 3b, 4b, f, j and Supplementary Figs. 6a, 7 are also available in Supplementary Fig. 10. The primary sequencing data and summary data files underlying Fig. 1, Tables 1, 2 and Supplementary Tables 1, 2 have been preserved (B.C.K. ORCID 0000-0002-2252-7634) as a DOI minted by Synapse.org: 10.7303/syn53060779.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-024-06570-2.

References

1.Ciechanover, A. Intracellular protein degradation: from a vague idea through the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting. Bioorg. Med. Chem.21, 3400–3410 (2013). 10.1016/j.bmc.2013.01.056 [DOI] [PubMed] [Google Scholar]
2.Yoshida, H., Haze, K., Yanagi, H., Yura, T. & Mori, K. Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins. Involvement of basic leucine zipper transcription factors. J. Biol. Chem.273, 33741–33749 (1998). 10.1074/jbc.273.50.33741 [DOI] [PubMed] [Google Scholar]
3.Roy, B. & Lee, A. S. The mammalian endoplasmic reticulum stress response element consists of an evolutionarily conserved tripartite structure and interacts with a novel stress-inducible complex. Nucleic Acids Res.27, 1437–1443 (1999). 10.1093/nar/27.6.1437 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Yoshida, H., Matsui, T., Yamamoto, A., Okada, T. & Mori, K. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell107, 881–891 (2001). 10.1016/S0092-8674(01)00611-0 [DOI] [PubMed] [Google Scholar]
5.Patil, C. & Walter, P. Intracellular signaling from the endoplasmic reticulum to the nucleus: the unfolded protein response in yeast and mammals. Curr. Opin. Cell Biol.13, 349–355 (2001). 10.1016/S0955-0674(00)00219-2 [DOI] [PubMed] [Google Scholar]
6.Haze, K., Yoshida, H., Yanagi, H., Yura, T. & Mori, K. Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress. Mol. Biol. Cell10, 3787–3799 (1999). 10.1091/mbc.10.11.3787 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Haze, K. et al. Identification of the G13 (cAMP-response-element-binding protein-related protein) gene product related to activating transcription factor 6 as a transcriptional activator of the mammalian unfolded protein response. Biochem. J.355, 19–28 (2001). 10.1042/bj3550019 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Ye, J. et al. ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Mol. Cell6, 1355–1364 (2000). 10.1016/S1097-2765(00)00133-7 [DOI] [PubMed] [Google Scholar]
9.Shen, J., Chen, X., Hendershot, L. & Prywes, R. ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals. Dev. Cell3, 99–111 (2002). 10.1016/S1534-5807(02)00203-4 [DOI] [PubMed] [Google Scholar]
10.Yoshida, H. et al. ATF6 activated by proteolysis binds in the presence of NF-Y (CBF) directly to the cis-acting element responsible for the mammalian unfolded protein response. Mol. Cell Biol.20, 6755–6767 (2000). 10.1128/MCB.20.18.6755-6767.2000 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Yoshida, H. et al. Endoplasmic reticulum stress-induced formation of transcription factor complex ERSF including NF-Y (CBF) and activating transcription factors 6alpha and 6beta that activates the mammalian unfolded protein response. Mol. Cell Biol.21, 1239–1248 (2001). 10.1128/MCB.21.4.1239-1248.2001 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Yoshida, H. et al. A time-dependent phase shift in the mammalian unfolded protein response. Dev. Cell4, 265–271 (2003). 10.1016/S1534-5807(03)00022-4 [DOI] [PubMed] [Google Scholar]
13.Yamamoto, K. et al. Transcriptional induction of mammalian ER quality control proteins is mediated by single or combined action of ATF6alpha and XBP1. Dev. Cell13, 365–376 (2007). 10.1016/j.devcel.2007.07.018 [DOI] [PubMed] [Google Scholar]
14.Ma, Y., Brewer, J. W., Diehl, J. A. & Hendershot, L. M. Two distinct stress signaling pathways converge upon the CHOP promoter during the mammalian unfolded protein response. J. Mol. Biol.318, 1351–1365 (2002). 10.1016/S0022-2836(02)00234-6 [DOI] [PubMed] [Google Scholar]
15.Lee, S., Sato, Y. & Nixon, R. A. Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer’s-like axonal dystrophy. J. Neurosci.31, 7817–7830 (2011). 10.1523/JNEUROSCI.6412-10.2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Ciechanover, A. & Kwon, Y. T. Degradation of misfolded proteins in neurodegenerative diseases: therapeutic targets and strategies. Exp. Mol. Med.47, e147 (2015). 10.1038/emm.2014.117 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Demuro, A. et al. Calcium dysregulation and membrane disruption as a ubiquitous neurotoxic mechanism of soluble amyloid oligomers. J. Biol. Chem.280, 17294–17300 (2005). 10.1074/jbc.M500997200 [DOI] [PubMed] [Google Scholar]
18.Ling, H. Untangling the tauopathies: current concepts of tau pathology and neurodegeneration. Parkinsonism Relat. Disord.46, S34–S38 (2018). 10.1016/j.parkreldis.2017.07.031 [DOI] [PubMed] [Google Scholar]
19.Guo, T., Noble, W. & Hanger, D. P. Roles of tau protein in health and disease. Acta Neuropathol.133, 665–704 (2017). 10.1007/s00401-017-1707-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Lee, V. M., Goedert, M. & Trojanowski, J. Q. Neurodegenerative tauopathies. Annu. Rev. Neurosci.24, 1121–1159 (2001). 10.1146/annurev.neuro.24.1.1121 [DOI] [PubMed] [Google Scholar]
21.Scheltens, P. et al. Alzheimer’s disease. Lancet397, 1577–1590 (2021). 10.1016/S0140-6736(20)32205-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Poorkaj, P. et al. Tau is a candidate gene for chromosome 17 frontotemporal dementia. Ann. Neurol.43, 815–825 (1998). 10.1002/ana.410430617 [DOI] [PubMed] [Google Scholar]
23.Hutton, M. et al. Association of missense and 5’-splice-site mutations in tau with the inherited dementia FTDP-17. Nature393, 702–705 (1998). 10.1038/31508 [DOI] [PubMed] [Google Scholar]
24.Spillantini, M. G. et al. Mutation in the tau gene in familial multiple system tauopathy with presenile dementia. Proc. Natl Acad. Sci. USA95, 7737–7741 (1998). 10.1073/pnas.95.13.7737 [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Hoozemans, J. J. et al. The unfolded protein response is activated in Alzheimer’s disease. Acta Neuropathol.110, 165–172 (2005). 10.1007/s00401-005-1038-0 [DOI] [PubMed] [Google Scholar]
26.Hoozemans, J. J. et al. The unfolded protein response is activated in pretangle neurons in Alzheimer’s disease hippocampus. Am. J. Pathol.174, 1241–1251 (2009). 10.2353/ajpath.2009.080814 [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Nijholt, D. A., van Haastert, E. S., Rozemuller, A. J., Scheper, W. & Hoozemans, J. J. The unfolded protein response is associated with early tau pathology in the hippocampus of tauopathies. J. Pathol.226, 693–702 (2012). 10.1002/path.3969 [DOI] [PubMed] [Google Scholar]
28.Stutzbach, L. D. et al. The unfolded protein response is activated in disease-affected brain regions in progressive supranuclear palsy and Alzheimer’s disease. Acta Neuropathol. Commun.1, 31 (2013). 10.1186/2051-5960-1-31 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Pitera, A. P. et al. Molecular investigation of the unfolded protein response in select human tauopathies. J. Alzheimers Dis. Rep.5, 855–869 (2021). 10.3233/ADR-210050 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Waldherr, S. M., Strovas, T. J., Vadset, T. A., Liachko, N. F. & Kraemer, B. C. Constitutive XBP-1s-mediated activation of the endoplasmic reticulum unfolded protein response protects against pathological tau. Nat. Commun.10, 4443 (2019). 10.1038/s41467-019-12070-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Taylor, L. M. et al. Pathological phosphorylation of tau and TDP-43 by TTBK1 and TTBK2 drives neurodegeneration. Mol. Neurodegener.13, 7 (2018). 10.1186/s13024-018-0237-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Taylor, R. C. & Dillin, A. XBP-1 is a cell-nonautonomous regulator of stress resistance and longevity. Cell153, 1435–1447 (2013). 10.1016/j.cell.2013.05.042 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Kawahara, T., Yanagi, H., Yura, T. & Mori, K. Endoplasmic reticulum stress-induced mRNA splicing permits synthesis of transcription factor Hac1p/Ern4p that activates the unfolded protein response. Mol. Biol. Cell8, 1845–1862 (1997). 10.1091/mbc.8.10.1845 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Cox, J. S., Chapman, R. E. & Walter, P. The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane. Mol. Biol. Cell8, 1805–1814 (1997). 10.1091/mbc.8.9.1805 [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Han, M. et al. Transgenic Dendra2::tau expression allows in vivo monitoring of tau proteostasis in Caenorhabditis elegans. Dis. Model. Mech.1710.1242/dmm.050473 (2024). [DOI] [PMC free article] [PubMed]
36.Kraemer, B. C. et al. Neurodegeneration and defective neurotransmission in a Caenorhabditis elegans model of tauopathy. Proc. Natl Acad. Sci. USA100, 9980–9985 (2003). 10.1073/pnas.1533448100 [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Riddle, D. L., Blumenthal, T., Meyer, B. J. & Priess, J. R. In C. elegans II (eds D. L. Riddle, T. Blumenthal, B. J. Meyer, & J. R. Priess). 2nd edn, (Cold Spring Harbor Laboratory Press, 1997). [PubMed]
38.Shen, X., Ellis, R. E., Sakaki, K. & Kaufman, R. J. Genetic interactions due to constitutive and inducible gene regulation mediated by the unfolded protein response in C. elegans. PLoS Genet.1, e37 (2005). 10.1371/journal.pgen.0010037 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Shoulders, M. D. et al. Stress-independent activation of XBP1s and/or ATF6 reveals three functionally diverse ER proteostasis environments. Cell Rep.3, 1279–1292 (2013). 10.1016/j.celrep.2013.03.024 [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Courreges, A. P., Najenson, A. C., Vatta, M. S. & Bianciotti, L. G. Atrial natriuretic peptide attenuates endoplasmic reticulum stress in experimental acute pancreatitis. Biochim. Biophys. Acta Mol. Basis Dis.1865, 485–493 (2019). 10.1016/j.bbadis.2018.12.004 [DOI] [PubMed] [Google Scholar]
41.van Huizen, R., Martindale, J. L., Gorospe, M. & Holbrook, N. J. P58IPK, a novel endoplasmic reticulum stress-inducible protein and potential negative regulator of eIF2alpha signaling. J. Biol. Chem.278, 15558–15564 (2003). 10.1074/jbc.M212074200 [DOI] [PubMed] [Google Scholar]
42.Sommer, T. & Jarosch, E. BiP binding keeps ATF6 at bay. Dev. Cell3, 1–2 (2002). 10.1016/S1534-5807(02)00210-1 [DOI] [PubMed] [Google Scholar]
43.Bommiasamy, H. et al. ATF6alpha induces XBP1-independent expansion of the endoplasmic reticulum. J. Cell Sci.122, 1626–1636 (2009). 10.1242/jcs.045625 [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Mao, H. Z. et al. Lipase maturation factor 1 (lmf1) is induced by endoplasmic reticulum stress through activating transcription factor 6alpha (Atf6alpha) signaling. J. Biol. Chem.289, 24417–24427 (2014). 10.1074/jbc.M114.588764 [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Kraemer, B. C., Burgess, J. K., Chen, J. H., Thomas, J. H. & Schellenberg, G. D. Molecular pathways that influence human tau-induced pathology in Caenorhabditis elegans. Hum. Mol. Genet.15, 1483–1496 (2006). 10.1093/hmg/ddl067 [DOI] [PubMed] [Google Scholar]
46.Eck, R. J., Kow, R. L., Black, A. H., Liachko, N. F. & Kraemer, B. C. SPOP loss of function protects against tauopathy. Proc. Natl Acad. Sci. USA120, e2207250120 (2023). 10.1073/pnas.2207250120 [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Kow, R. L., Black, A. H., Henderson, B. P. & Kraemer, B. C. Sut-6/NIPP1 modulates tau toxicity. Hum. Mol. Genet.32, 2292–2306 (2023). 10.1093/hmg/ddad049 [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Latimer, C. S. et al. TDP-43 promotes tau accumulation and selective neurotoxicity in bigenic Caenorhabditis elegans. Dis. Model Mech. 1510.1242/dmm.049323 (2022). [DOI] [PMC free article] [PubMed]
49.Kow, R. L., Black, A. H., Saxton, A. D., Liachko, N. F. & Kraemer, B. C. Loss of aly/ALYREF suppresses toxicity in both tau and TDP-43 models of neurodegeneration. Geroscience44, 747–761 (2022). 10.1007/s11357-022-00526-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Kow, R. L. et al. Distinct Poly(A) nucleases have differential impact on sut-2 dependent tauopathy phenotypes. Neurobiol. Dis.147, 105148 (2021). 10.1016/j.nbd.2020.105148 [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Baker, J. D., Uhrich, R. L., Strovas, T. J., Saxton, A. D. & Kraemer, B. C. Targeting pathological tau by small molecule inhibition of the Poly(A):MSUT2 RNA-protein interaction. ACS Chem. Neurosci.11, 2277–2285 (2020). 10.1021/acschemneuro.0c00214 [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Latimer, C. S. et al. Resistance and resilience to Alzheimer’s disease pathology are associated with reduced cortical pTau and absence of limbic-predominant age-related TDP-43 encephalopathy in a community-based cohort. Acta Neuropathol. Commun.7, 91 (2019). 10.1186/s40478-019-0743-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Kow, R. L., Sikkema, C., Wheeler, J. M., Wilkinson, C. W. & Kraemer, B. C. DOPA decarboxylase modulates tau toxicity. Biol. Psychiatry83, 438–446 (2018). 10.1016/j.biopsych.2017.06.007 [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Liachko, N. F. et al. The tau tubulin kinases TTBK1/2 promote accumulation of pathological TDP-43. PLoS Genet.10, e1004803 (2014). 10.1371/journal.pgen.1004803 [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Liachko, N. F. et al. CDC7 inhibition blocks pathological TDP-43 phosphorylation and neurodegeneration. Ann. Neurol.74, 39–52 (2013). 10.1002/ana.23870 [DOI] [PMC free article] [PubMed] [Google Scholar]
56.McCormick, A. V., Wheeler, J. M., Guthrie, C. R., Liachko, N. F. & Kraemer, B. C. Dopamine D2 receptor antagonism suppresses tau aggregation and neurotoxicity. Biol. Psychiatry73, 464–471 (2013). 10.1016/j.biopsych.2012.08.027 [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Guthrie, C. R., Greenup, L., Leverenz, J. B. & Kraemer, B. C. MSUT2 is a determinant of susceptibility to tau neurotoxicity. Hum. Mol. Genet.20, 1989–1999 (2011). 10.1093/hmg/ddr079 [DOI] [PMC free article] [PubMed] [Google Scholar]
58.Liachko, N. F., Guthrie, C. R. & Kraemer, B. C. Phosphorylation promotes neurotoxicity in a Caenorhabditis elegans model of TDP-43 proteinopathy. J. Neurosci.30, 16208–16219 (2010). 10.1523/JNEUROSCI.2911-10.2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Guthrie, C. R., Schellenberg, G. D. & Kraemer, B. C. SUT-2 potentiates tau-induced neurotoxicity in Caenorhabditis elegans. Hum. Mol. Genet18, 1825–1838 (2009). 10.1093/hmg/ddp099 [DOI] [PMC free article] [PubMed] [Google Scholar]
60.Sternberg, P. W. et al. WormBase 2024: status and transitioning to Alliance infrastructure. Genetics 227 10.1093/genetics/iyae050 (2024). [DOI] [PMC free article] [PubMed]
61.Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M. & Horvitz, H. R. The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. Cell75, 641–652 (1993). 10.1016/0092-8674(93)90485-9 [DOI] [PubMed] [Google Scholar]
62.Fan, T. J., Han, L. H., Cong, R. S. & Liang, J. Caspase family proteases and apoptosis. Acta Biochim. Biophys. Sin.37, 719–727 (2005). 10.1111/j.1745-7270.2005.00108.x [DOI] [PubMed] [Google Scholar]
63.Shi, Y. Mechanisms of caspase activation and inhibition during apoptosis. Mol. Cell9, 459–470 (2002). 10.1016/S1097-2765(02)00482-3 [DOI] [PubMed] [Google Scholar]
64.Denning, D. P., Hatch, V. & Horvitz, H. R. Both the caspase CSP-1 and a caspase-independent pathway promote programmed cell death in parallel to the canonical pathway for apoptosis in Caenorhabditis elegans. PLoS Genet.9, e1003341 (2013). 10.1371/journal.pgen.1003341 [DOI] [PMC free article] [PubMed] [Google Scholar]
65.Yan, W. et al. Control of PERK eIF2alpha kinase activity by the endoplasmic reticulum stress-induced molecular chaperone P58IPK. Proc. Natl Acad. Sci. USA99, 15920–15925 (2002). 10.1073/pnas.252341799 [DOI] [PMC free article] [PubMed] [Google Scholar]
66.Otero, J. H., Lizak, B. & Hendershot, L. M. Life and death of a BiP substrate. Semin. Cell Dev. Biol.21, 472–478 (2010). 10.1016/j.semcdb.2009.12.008 [DOI] [PMC free article] [PubMed] [Google Scholar]
67.Rutkowski, D. T. et al. The role of p58IPK in protecting the stressed endoplasmic reticulum. Mol. Biol. Cell18, 3681–3691 (2007). 10.1091/mbc.e07-03-0272 [DOI] [PMC free article] [PubMed] [Google Scholar]
68.Petrova, K., Oyadomari, S., Hendershot, L. M. & Ron, D. Regulated association of misfolded endoplasmic reticulum lumenal proteins with P58/DNAJc3. EMBO J.27, 2862–2872 (2008). 10.1038/emboj.2008.199 [DOI] [PMC free article] [PubMed] [Google Scholar]
69.Tao, J., Wu, Y., Ron, D. & Sha, B. Preliminary X-ray crystallographic studies of mouse UPR responsive protein P58(IPK) TPR fragment. Acta Crystallogr. Sect. F. Struct. Biol. Cryst. Commun.64, 108–110 (2008). 10.1107/S1744309108000833 [DOI] [PMC free article] [PubMed] [Google Scholar]
70.Munoz-Lobato, F. et al. Protective role of DNJ-27/ERdj5 in Caenorhabditis elegans models of human neurodegenerative diseases. Antioxid. Redox Signal20, 217–235 (2014). 10.1089/ars.2012.5051 [DOI] [PMC free article] [PubMed] [Google Scholar]
71.Wang, J., Lee, J., Liem, D. & Ping, P. HSPA5 Gene encoding Hsp70 chaperone BiP in the endoplasmic reticulum. Gene618, 14–23 (2017). 10.1016/j.gene.2017.03.005 [DOI] [PMC free article] [PubMed] [Google Scholar]
72.Shen, X. et al. Complementary signaling pathways regulate the unfolded protein response and are required for C. elegans development. Cell107, 893–903 (2001). 10.1016/S0092-8674(01)00612-2 [DOI] [PubMed] [Google Scholar]
73.Kapulkin, W. J., Hiester, B. G. & Link, C. D. Compensatory regulation among ER chaperones in C. elegans. FEBS Lett.579, 3063–3068 (2005). 10.1016/j.febslet.2005.04.062 [DOI] [PubMed] [Google Scholar]
74.Sriburi, R. et al. Coordinate regulation of phospholipid biosynthesis and secretory pathway gene expression in XBP-1(S)-induced endoplasmic reticulum biogenesis. J. Biol. Chem.282, 7024–7034 (2007). 10.1074/jbc.M609490200 [DOI] [PubMed] [Google Scholar]
75.Li, F. & Zhang, H. Lysosomal acid lipase in lipid metabolism and beyond. Arterioscler. Thromb. Vasc. Biol.39, 850–856 (2019). 10.1161/ATVBAHA.119.312136 [DOI] [PMC free article] [PubMed] [Google Scholar]
76.Tempio, T. et al. A virtuous cycle operated by ERp44 and ERGIC-53 guarantees proteostasis in the early secretory compartment. iScience24, 102244 (2021). 10.1016/j.isci.2021.102244 [DOI] [PMC free article] [PubMed] [Google Scholar]
77.Le Borgne, F., Ben Mohamed, A., Logerot, M., Garnier, E. & Demarquoy, J. Changes in carnitine octanoyltransferase activity induce alteration in fatty acid metabolism. Biochem. Biophys. Res. Commun.409, 699–704 (2011). 10.1016/j.bbrc.2011.05.068 [DOI] [PubMed] [Google Scholar]
78.Timson, D. J. & Reece, R. J. Identification and characterisation of human aldose 1-epimerase. FEBS Lett.543, 21–24 (2003). 10.1016/S0014-5793(03)00364-8 [DOI] [PubMed] [Google Scholar]
79.Jiao, X., Chang, J. H., Kilic, T., Tong, L. & Kiledjian, M. A mammalian pre-mRNA 5’ end capping quality control mechanism and an unexpected link of capping to pre-mRNA processing. Mol. Cell50, 104–115 (2013). 10.1016/j.molcel.2013.02.017 [DOI] [PMC free article] [PubMed] [Google Scholar]
80.Doamekpor, S. K. et al. DXO/Rai1 enzymes remove 5’-end FAD and dephospho-CoA caps on RNAs. Nucleic Acids Res.48, 6136–6148 (2020). 10.1093/nar/gkaa297 [DOI] [PMC free article] [PubMed] [Google Scholar]
81.Grudzien-Nogalska, E. & Kiledjian, M. New insights into decapping enzymes and selective mRNA decay. Wiley Interdiscip. Rev. RNA810.1002/wrna.1379 (2017). [DOI] [PMC free article] [PubMed]
82.Stelzer, G. et al. The GeneCards Suite: From Gene Data Mining to Disease Genome Sequence Analyses. Curr Protoc Bioinformatics. 54, 10.1002/cpbi.5 (2016). [DOI] [PubMed]
83.Sayers, E. W. et al. Database resources of the national center for biotechnology information. Nucleic Acids Res. 50, D20-D26 10.1093/nar/gkab1112 (2022). [DOI] [PMC free article] [PubMed]
84.Halestrap, A. P. The monocarboxylate transporter family–structure and functional characterization. IUBMB Life64, 1–9 (2012). 10.1002/iub.573 [DOI] [PubMed] [Google Scholar]
85.Deng, Y. et al. The Xbp1s/GalE axis links ER stress to postprandial hepatic metabolism. J. Clin. Invest.123, 455–468 (2013). 10.1172/JCI62819 [DOI] [PMC free article] [PubMed] [Google Scholar]
86.Huang, C., Tong, J., Bi, F., Zhou, H. & Xia, X. G. Mutant TDP-43 in motor neurons promotes the onset and progression of ALS in rats. J. Clin. Invest.122, 107–118 (2012). 10.1172/JCI59130 [DOI] [PMC free article] [PubMed] [Google Scholar]
87.Walker, A. K. et al. Functional recovery in new mouse models of ALS/FTLD after clearance of pathological cytoplasmic TDP-43. Acta Neuropathol.130, 643–660 (2015). 10.1007/s00401-015-1460-x [DOI] [PMC free article] [PubMed] [Google Scholar]
88.Marciniak, S. J. et al. CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum. Genes Dev.18, 3066–3077 (2004). 10.1101/gad.1250704 [DOI] [PMC free article] [PubMed] [Google Scholar]
89.Urra, H., Dufey, E., Lisbona, F., Rojas-Rivera, D. & Hetz, C. When ER stress reaches a dead end. Biochim. Biophys. Acta1833, 3507–3517 (2013). 10.1016/j.bbamcr.2013.07.024 [DOI] [PubMed] [Google Scholar]
90.Imanikia, S., Ozbey, N. P., Krueger, C., Casanueva, M. O. & Taylor, R. C. Neuronal XBP-1 activates intestinal lysosomes to improve proteostasis in C. elegans. Curr. Biol.29, 2322–2338.e2327 (2019). 10.1016/j.cub.2019.06.031 [DOI] [PMC free article] [PubMed] [Google Scholar]
91.Zarouchlioti, C., Parfitt, D. A., Li, W., Gittings, L. M. & Cheetham, M. E. DNAJ proteins in neurodegeneration: essential and protective factors. Philos. Trans. R. Soc. Lond. B Biol. Sci.37310.1098/rstb.2016.0534 (2018). [DOI] [PMC free article] [PubMed]
92.Damiano, F., Testini, M., Tocci, R., Gnoni, G. V. & Siculella, L. Translational control of human acetyl-CoA carboxylase 1 mRNA is mediated by an internal ribosome entry site in response to ER stress, serum deprivation or hypoxia mimetic CoCl(2). Biochim. Biophys. Acta Mol. Cell Biol. Lipids1863, 388–398 (2018). 10.1016/j.bbalip.2018.01.006 [DOI] [PubMed] [Google Scholar]
93.Morrison, J. K., Friday, A. J., Henderson, M. A., Hao, E. & Keiper, B. D. Induction of cap-independent BiP (hsp-3) and Bcl-2 (ced-9) translation in response to eIF4G (IFG-1) depletion in C. elegans. Translation2, e28935 (2014). 10.4161/trla.28935 [DOI] [PMC free article] [PubMed] [Google Scholar]
94.Imanikia, S., Sheng, M., Castro, C., Griffin, J. L. & Taylor, R. C. XBP-1 remodels lipid metabolism to extend longevity. Cell Rep.28, 581–589.e584 (2019). 10.1016/j.celrep.2019.06.057 [DOI] [PMC free article] [PubMed] [Google Scholar]
95.Daniele, J. R. et al. UPR(ER) promotes lipophagy independent of chaperones to extend life span. Sci. Adv.6, eaaz1441 (2020). 10.1126/sciadv.aaz1441 [DOI] [PMC free article] [PubMed] [Google Scholar]
96.Ho, N. et al. Stress sensor Ire1 deploys a divergent transcriptional program in response to lipid bilayer stress. J. Cell Biol.21910.1083/jcb.201909165 (2020). [DOI] [PMC free article] [PubMed]
97.Piperi, C., Adamopoulos, C. & Papavassiliou, A. G. XBP1: a pivotal transcriptional regulator of glucose and lipid metabolism. Trends Endocrinol. Metab.27, 119–122 (2016). 10.1016/j.tem.2016.01.001 [DOI] [PubMed] [Google Scholar]
98.Ozbey, N. P. et al. Tyramine acts downstream of neuronal XBP-1s to coordinate inter-tissue UPR(ER) activation and behavior in C. elegans. Dev. Cell55, 754–770.e756 (2020). 10.1016/j.devcel.2020.10.024 [DOI] [PMC free article] [PubMed] [Google Scholar]
99.Ellis, H. M. & Horvitz, H. R. Genetic control of programmed cell death in the nematode C. elegans. Cell44, 817–829 (1986). 10.1016/0092-8674(86)90004-8 [DOI] [PubMed] [Google Scholar]
100.Shaham, S. Identification of multiple Caenorhabditis elegans caspases and their potential roles in proteolytic cascades. J. Biol. Chem.273, 35109–35117 (1998). 10.1074/jbc.273.52.35109 [DOI] [PubMed] [Google Scholar]
101.Slee, E. A., Adrain, C. & Martin, S. J. Executioner caspase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis. J. Biol. Chem.276, 7320–7326 (2001). 10.1074/jbc.M008363200 [DOI] [PubMed] [Google Scholar]
102.Hitomi, J. et al. Apoptosis induced by endoplasmic reticulum stress depends on activation of caspase-3 via caspase-12. Neurosci. Lett.357, 127–130 (2004). 10.1016/j.neulet.2003.12.080 [DOI] [PubMed] [Google Scholar]
103.Kaufman, R. J. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev.13, 1211–1233 (1999). 10.1101/gad.13.10.1211 [DOI] [PubMed] [Google Scholar]
104.Guo, H. et al. Active caspase-6 and caspase-6-cleaved tau in neuropil threads, neuritic plaques, and neurofibrillary tangles of Alzheimer’s disease. Am. J. Pathol.165, 523–531 (2004). 10.1016/S0002-9440(10)63317-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
105.Selznick, L. A. et al. In situ immunodetection of neuronal caspase-3 activation in Alzheimer disease. J. Neuropathol. Exp. Neurol.58, 1020–1026 (1999). 10.1097/00005072-199909000-00012 [DOI] [PubMed] [Google Scholar]
106.Wai, M. S. et al. Co-localization of hyperphosphorylated tau and caspases in the brainstem of Alzheimer’s disease patients. Biogerontology10, 457–469 (2009). 10.1007/s10522-008-9189-8 [DOI] [PubMed] [Google Scholar]
107.Wang, Z. et al. Sustained overexpression of spliced X-box-binding protein-1 in neurons leads to spontaneous seizures and sudden death in mice. Commun. Biol.6, 252 (2023). 10.1038/s42003-023-04594-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
108.Martinez, G. et al. Regulation of memory formation by the transcription factor XBP1. Cell Rep.14, 1382–1394 (2016). 10.1016/j.celrep.2016.01.028 [DOI] [PubMed] [Google Scholar]
109.Duran-Aniotz, C. et al. The unfolded protein response transcription factor XBP1s ameliorates Alzheimer’s disease by improving synaptic function and proteostasis. Mol. Ther.31, 2240–2256 (2023). 10.1016/j.ymthe.2023.03.028 [DOI] [PMC free article] [PubMed] [Google Scholar]
110.Cisse, M. et al. The transcription factor XBP1s restores hippocampal synaptic plasticity and memory by control of the Kalirin-7 pathway in Alzheimer model. Mol. Psychiatry22, 1562–1575 (2017). 10.1038/mp.2016.152 [DOI] [PMC free article] [PubMed] [Google Scholar]
111.Chatterjee, S. et al. Endoplasmic reticulum chaperone genes encode effectors of long-term memory. Sci. Adv.8, eabm6063 (2022). 10.1126/sciadv.abm6063 [DOI] [PMC free article] [PubMed] [Google Scholar]
112.Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods6, 343–345 (2009). 10.1038/nmeth.1318 [DOI] [PubMed] [Google Scholar]
113.Brenner, S. The genetics of Caenorhabditis elegans. Genetics77, 71–94 (1974). 10.1093/genetics/77.1.71 [DOI] [PMC free article] [PubMed] [Google Scholar]
114.American Society for Cell Biology, Epstein, H. F. & Shakes, D. C. in Methods in Cell Biology, 1 online resource (xxi) 659 (Academic Press, 1995).
115.Wood, W. B. The Nematode Caenorhabditis elegans (Cold Spring Harbor Laboratory, 1988).
116.Paix, A., Folkmann, A., Rasoloson, D. & Seydoux, G. High efficiency, homology-directed genome editing in Caenorhabditis elegans using CRISPR-Cas9 ribonucleoprotein complexes. Genetics201, 47–54 (2015). 10.1534/genetics.115.179382 [DOI] [PMC free article] [PubMed] [Google Scholar]
117.Currey, H. N. & Liachko, N. F. Evaluation of motor impairment in C. elegans models of amyotrophic lateral sclerosis. J. Vis. Exp.10.3791/62699 (2021). [DOI] [PubMed]
118.Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH image to ImageJ: 25 years of image analysis. Nat. Methods9, 671–675 (2012). 10.1038/nmeth.2089 [DOI] [PMC free article] [PubMed] [Google Scholar]
119.Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol.215, 403–410 (1990). 10.1016/S0022-2836(05)80360-2 [DOI] [PubMed] [Google Scholar]
120.Sherman, B. T. et al. DAVID: a web server for functional enrichment analysis and functional annotation of gene lists (2021 update). Nucleic Acids Res.50, W216–W221 (2022). 10.1093/nar/gkac194 [DOI] [PMC free article] [PubMed] [Google Scholar]
121.Tang, D. et al. SRplot: a free online platform for data visualization and graphing. PLoS ONE18, e0294236 (2023). 10.1371/journal.pone.0294236 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Information^{(1.3MB, pdf)}

42003_2024_6570_MOESM2_ESM.pdf^{(26.4KB, pdf)}

Description of Additional Supplementary Files

Supplementary Data 1^{(1.2MB, xlsx)}

Reporting Summary^{(2.1MB, pdf)}

Data Availability Statement

[CR1] 1.Ciechanover, A. Intracellular protein degradation: from a vague idea through the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting. Bioorg. Med. Chem.21, 3400–3410 (2013). 10.1016/j.bmc.2013.01.056 [DOI] [PubMed] [Google Scholar]

[CR2] 2.Yoshida, H., Haze, K., Yanagi, H., Yura, T. & Mori, K. Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins. Involvement of basic leucine zipper transcription factors. J. Biol. Chem.273, 33741–33749 (1998). 10.1074/jbc.273.50.33741 [DOI] [PubMed] [Google Scholar]

[CR3] 3.Roy, B. & Lee, A. S. The mammalian endoplasmic reticulum stress response element consists of an evolutionarily conserved tripartite structure and interacts with a novel stress-inducible complex. Nucleic Acids Res.27, 1437–1443 (1999). 10.1093/nar/27.6.1437 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR4] 4.Yoshida, H., Matsui, T., Yamamoto, A., Okada, T. & Mori, K. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell107, 881–891 (2001). 10.1016/S0092-8674(01)00611-0 [DOI] [PubMed] [Google Scholar]

[CR5] 5.Patil, C. & Walter, P. Intracellular signaling from the endoplasmic reticulum to the nucleus: the unfolded protein response in yeast and mammals. Curr. Opin. Cell Biol.13, 349–355 (2001). 10.1016/S0955-0674(00)00219-2 [DOI] [PubMed] [Google Scholar]

[CR6] 6.Haze, K., Yoshida, H., Yanagi, H., Yura, T. & Mori, K. Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress. Mol. Biol. Cell10, 3787–3799 (1999). 10.1091/mbc.10.11.3787 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR7] 7.Haze, K. et al. Identification of the G13 (cAMP-response-element-binding protein-related protein) gene product related to activating transcription factor 6 as a transcriptional activator of the mammalian unfolded protein response. Biochem. J.355, 19–28 (2001). 10.1042/bj3550019 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR8] 8.Ye, J. et al. ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Mol. Cell6, 1355–1364 (2000). 10.1016/S1097-2765(00)00133-7 [DOI] [PubMed] [Google Scholar]

[CR9] 9.Shen, J., Chen, X., Hendershot, L. & Prywes, R. ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals. Dev. Cell3, 99–111 (2002). 10.1016/S1534-5807(02)00203-4 [DOI] [PubMed] [Google Scholar]

[CR10] 10.Yoshida, H. et al. ATF6 activated by proteolysis binds in the presence of NF-Y (CBF) directly to the cis-acting element responsible for the mammalian unfolded protein response. Mol. Cell Biol.20, 6755–6767 (2000). 10.1128/MCB.20.18.6755-6767.2000 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR11] 11.Yoshida, H. et al. Endoplasmic reticulum stress-induced formation of transcription factor complex ERSF including NF-Y (CBF) and activating transcription factors 6alpha and 6beta that activates the mammalian unfolded protein response. Mol. Cell Biol.21, 1239–1248 (2001). 10.1128/MCB.21.4.1239-1248.2001 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR12] 12.Yoshida, H. et al. A time-dependent phase shift in the mammalian unfolded protein response. Dev. Cell4, 265–271 (2003). 10.1016/S1534-5807(03)00022-4 [DOI] [PubMed] [Google Scholar]

[CR13] 13.Yamamoto, K. et al. Transcriptional induction of mammalian ER quality control proteins is mediated by single or combined action of ATF6alpha and XBP1. Dev. Cell13, 365–376 (2007). 10.1016/j.devcel.2007.07.018 [DOI] [PubMed] [Google Scholar]

[CR14] 14.Ma, Y., Brewer, J. W., Diehl, J. A. & Hendershot, L. M. Two distinct stress signaling pathways converge upon the CHOP promoter during the mammalian unfolded protein response. J. Mol. Biol.318, 1351–1365 (2002). 10.1016/S0022-2836(02)00234-6 [DOI] [PubMed] [Google Scholar]

[CR15] 15.Lee, S., Sato, Y. & Nixon, R. A. Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer’s-like axonal dystrophy. J. Neurosci.31, 7817–7830 (2011). 10.1523/JNEUROSCI.6412-10.2011 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR16] 16.Ciechanover, A. & Kwon, Y. T. Degradation of misfolded proteins in neurodegenerative diseases: therapeutic targets and strategies. Exp. Mol. Med.47, e147 (2015). 10.1038/emm.2014.117 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR17] 17.Demuro, A. et al. Calcium dysregulation and membrane disruption as a ubiquitous neurotoxic mechanism of soluble amyloid oligomers. J. Biol. Chem.280, 17294–17300 (2005). 10.1074/jbc.M500997200 [DOI] [PubMed] [Google Scholar]

[CR18] 18.Ling, H. Untangling the tauopathies: current concepts of tau pathology and neurodegeneration. Parkinsonism Relat. Disord.46, S34–S38 (2018). 10.1016/j.parkreldis.2017.07.031 [DOI] [PubMed] [Google Scholar]

[CR19] 19.Guo, T., Noble, W. & Hanger, D. P. Roles of tau protein in health and disease. Acta Neuropathol.133, 665–704 (2017). 10.1007/s00401-017-1707-9 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR20] 20.Lee, V. M., Goedert, M. & Trojanowski, J. Q. Neurodegenerative tauopathies. Annu. Rev. Neurosci.24, 1121–1159 (2001). 10.1146/annurev.neuro.24.1.1121 [DOI] [PubMed] [Google Scholar]

[CR21] 21.Scheltens, P. et al. Alzheimer’s disease. Lancet397, 1577–1590 (2021). 10.1016/S0140-6736(20)32205-4 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR22] 22.Poorkaj, P. et al. Tau is a candidate gene for chromosome 17 frontotemporal dementia. Ann. Neurol.43, 815–825 (1998). 10.1002/ana.410430617 [DOI] [PubMed] [Google Scholar]

[CR23] 23.Hutton, M. et al. Association of missense and 5’-splice-site mutations in tau with the inherited dementia FTDP-17. Nature393, 702–705 (1998). 10.1038/31508 [DOI] [PubMed] [Google Scholar]

[CR24] 24.Spillantini, M. G. et al. Mutation in the tau gene in familial multiple system tauopathy with presenile dementia. Proc. Natl Acad. Sci. USA95, 7737–7741 (1998). 10.1073/pnas.95.13.7737 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR25] 25.Hoozemans, J. J. et al. The unfolded protein response is activated in Alzheimer’s disease. Acta Neuropathol.110, 165–172 (2005). 10.1007/s00401-005-1038-0 [DOI] [PubMed] [Google Scholar]

[CR26] 26.Hoozemans, J. J. et al. The unfolded protein response is activated in pretangle neurons in Alzheimer’s disease hippocampus. Am. J. Pathol.174, 1241–1251 (2009). 10.2353/ajpath.2009.080814 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR27] 27.Nijholt, D. A., van Haastert, E. S., Rozemuller, A. J., Scheper, W. & Hoozemans, J. J. The unfolded protein response is associated with early tau pathology in the hippocampus of tauopathies. J. Pathol.226, 693–702 (2012). 10.1002/path.3969 [DOI] [PubMed] [Google Scholar]

[CR28] 28.Stutzbach, L. D. et al. The unfolded protein response is activated in disease-affected brain regions in progressive supranuclear palsy and Alzheimer’s disease. Acta Neuropathol. Commun.1, 31 (2013). 10.1186/2051-5960-1-31 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR29] 29.Pitera, A. P. et al. Molecular investigation of the unfolded protein response in select human tauopathies. J. Alzheimers Dis. Rep.5, 855–869 (2021). 10.3233/ADR-210050 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR30] 30.Waldherr, S. M., Strovas, T. J., Vadset, T. A., Liachko, N. F. & Kraemer, B. C. Constitutive XBP-1s-mediated activation of the endoplasmic reticulum unfolded protein response protects against pathological tau. Nat. Commun.10, 4443 (2019). 10.1038/s41467-019-12070-3 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR31] 31.Taylor, L. M. et al. Pathological phosphorylation of tau and TDP-43 by TTBK1 and TTBK2 drives neurodegeneration. Mol. Neurodegener.13, 7 (2018). 10.1186/s13024-018-0237-9 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR32] 32.Taylor, R. C. & Dillin, A. XBP-1 is a cell-nonautonomous regulator of stress resistance and longevity. Cell153, 1435–1447 (2013). 10.1016/j.cell.2013.05.042 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR33] 33.Kawahara, T., Yanagi, H., Yura, T. & Mori, K. Endoplasmic reticulum stress-induced mRNA splicing permits synthesis of transcription factor Hac1p/Ern4p that activates the unfolded protein response. Mol. Biol. Cell8, 1845–1862 (1997). 10.1091/mbc.8.10.1845 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR34] 34.Cox, J. S., Chapman, R. E. & Walter, P. The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane. Mol. Biol. Cell8, 1805–1814 (1997). 10.1091/mbc.8.9.1805 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR35] 35.Han, M. et al. Transgenic Dendra2::tau expression allows in vivo monitoring of tau proteostasis in Caenorhabditis elegans. Dis. Model. Mech.1710.1242/dmm.050473 (2024). [DOI] [PMC free article] [PubMed]

[CR36] 36.Kraemer, B. C. et al. Neurodegeneration and defective neurotransmission in a Caenorhabditis elegans model of tauopathy. Proc. Natl Acad. Sci. USA100, 9980–9985 (2003). 10.1073/pnas.1533448100 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR37] 37.Riddle, D. L., Blumenthal, T., Meyer, B. J. & Priess, J. R. In C. elegans II (eds D. L. Riddle, T. Blumenthal, B. J. Meyer, & J. R. Priess). 2nd edn, (Cold Spring Harbor Laboratory Press, 1997). [PubMed]

[CR38] 38.Shen, X., Ellis, R. E., Sakaki, K. & Kaufman, R. J. Genetic interactions due to constitutive and inducible gene regulation mediated by the unfolded protein response in C. elegans. PLoS Genet.1, e37 (2005). 10.1371/journal.pgen.0010037 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR39] 39.Shoulders, M. D. et al. Stress-independent activation of XBP1s and/or ATF6 reveals three functionally diverse ER proteostasis environments. Cell Rep.3, 1279–1292 (2013). 10.1016/j.celrep.2013.03.024 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR40] 40.Courreges, A. P., Najenson, A. C., Vatta, M. S. & Bianciotti, L. G. Atrial natriuretic peptide attenuates endoplasmic reticulum stress in experimental acute pancreatitis. Biochim. Biophys. Acta Mol. Basis Dis.1865, 485–493 (2019). 10.1016/j.bbadis.2018.12.004 [DOI] [PubMed] [Google Scholar]

[CR41] 41.van Huizen, R., Martindale, J. L., Gorospe, M. & Holbrook, N. J. P58IPK, a novel endoplasmic reticulum stress-inducible protein and potential negative regulator of eIF2alpha signaling. J. Biol. Chem.278, 15558–15564 (2003). 10.1074/jbc.M212074200 [DOI] [PubMed] [Google Scholar]

[CR42] 42.Sommer, T. & Jarosch, E. BiP binding keeps ATF6 at bay. Dev. Cell3, 1–2 (2002). 10.1016/S1534-5807(02)00210-1 [DOI] [PubMed] [Google Scholar]

[CR43] 43.Bommiasamy, H. et al. ATF6alpha induces XBP1-independent expansion of the endoplasmic reticulum. J. Cell Sci.122, 1626–1636 (2009). 10.1242/jcs.045625 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR44] 44.Mao, H. Z. et al. Lipase maturation factor 1 (lmf1) is induced by endoplasmic reticulum stress through activating transcription factor 6alpha (Atf6alpha) signaling. J. Biol. Chem.289, 24417–24427 (2014). 10.1074/jbc.M114.588764 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR45] 45.Kraemer, B. C., Burgess, J. K., Chen, J. H., Thomas, J. H. & Schellenberg, G. D. Molecular pathways that influence human tau-induced pathology in Caenorhabditis elegans. Hum. Mol. Genet.15, 1483–1496 (2006). 10.1093/hmg/ddl067 [DOI] [PubMed] [Google Scholar]

[CR46] 46.Eck, R. J., Kow, R. L., Black, A. H., Liachko, N. F. & Kraemer, B. C. SPOP loss of function protects against tauopathy. Proc. Natl Acad. Sci. USA120, e2207250120 (2023). 10.1073/pnas.2207250120 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR47] 47.Kow, R. L., Black, A. H., Henderson, B. P. & Kraemer, B. C. Sut-6/NIPP1 modulates tau toxicity. Hum. Mol. Genet.32, 2292–2306 (2023). 10.1093/hmg/ddad049 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR48] 48.Latimer, C. S. et al. TDP-43 promotes tau accumulation and selective neurotoxicity in bigenic Caenorhabditis elegans. Dis. Model Mech. 1510.1242/dmm.049323 (2022). [DOI] [PMC free article] [PubMed]

[CR49] 49.Kow, R. L., Black, A. H., Saxton, A. D., Liachko, N. F. & Kraemer, B. C. Loss of aly/ALYREF suppresses toxicity in both tau and TDP-43 models of neurodegeneration. Geroscience44, 747–761 (2022). 10.1007/s11357-022-00526-2 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR50] 50.Kow, R. L. et al. Distinct Poly(A) nucleases have differential impact on sut-2 dependent tauopathy phenotypes. Neurobiol. Dis.147, 105148 (2021). 10.1016/j.nbd.2020.105148 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR51] 51.Baker, J. D., Uhrich, R. L., Strovas, T. J., Saxton, A. D. & Kraemer, B. C. Targeting pathological tau by small molecule inhibition of the Poly(A):MSUT2 RNA-protein interaction. ACS Chem. Neurosci.11, 2277–2285 (2020). 10.1021/acschemneuro.0c00214 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR52] 52.Latimer, C. S. et al. Resistance and resilience to Alzheimer’s disease pathology are associated with reduced cortical pTau and absence of limbic-predominant age-related TDP-43 encephalopathy in a community-based cohort. Acta Neuropathol. Commun.7, 91 (2019). 10.1186/s40478-019-0743-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR53] 53.Kow, R. L., Sikkema, C., Wheeler, J. M., Wilkinson, C. W. & Kraemer, B. C. DOPA decarboxylase modulates tau toxicity. Biol. Psychiatry83, 438–446 (2018). 10.1016/j.biopsych.2017.06.007 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR54] 54.Liachko, N. F. et al. The tau tubulin kinases TTBK1/2 promote accumulation of pathological TDP-43. PLoS Genet.10, e1004803 (2014). 10.1371/journal.pgen.1004803 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR55] 55.Liachko, N. F. et al. CDC7 inhibition blocks pathological TDP-43 phosphorylation and neurodegeneration. Ann. Neurol.74, 39–52 (2013). 10.1002/ana.23870 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR56] 56.McCormick, A. V., Wheeler, J. M., Guthrie, C. R., Liachko, N. F. & Kraemer, B. C. Dopamine D2 receptor antagonism suppresses tau aggregation and neurotoxicity. Biol. Psychiatry73, 464–471 (2013). 10.1016/j.biopsych.2012.08.027 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR57] 57.Guthrie, C. R., Greenup, L., Leverenz, J. B. & Kraemer, B. C. MSUT2 is a determinant of susceptibility to tau neurotoxicity. Hum. Mol. Genet.20, 1989–1999 (2011). 10.1093/hmg/ddr079 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR58] 58.Liachko, N. F., Guthrie, C. R. & Kraemer, B. C. Phosphorylation promotes neurotoxicity in a Caenorhabditis elegans model of TDP-43 proteinopathy. J. Neurosci.30, 16208–16219 (2010). 10.1523/JNEUROSCI.2911-10.2010 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR59] 59.Guthrie, C. R., Schellenberg, G. D. & Kraemer, B. C. SUT-2 potentiates tau-induced neurotoxicity in Caenorhabditis elegans. Hum. Mol. Genet18, 1825–1838 (2009). 10.1093/hmg/ddp099 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR60] 60.Sternberg, P. W. et al. WormBase 2024: status and transitioning to Alliance infrastructure. Genetics 227 10.1093/genetics/iyae050 (2024). [DOI] [PMC free article] [PubMed]

[CR61] 61.Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M. & Horvitz, H. R. The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. Cell75, 641–652 (1993). 10.1016/0092-8674(93)90485-9 [DOI] [PubMed] [Google Scholar]

[CR62] 62.Fan, T. J., Han, L. H., Cong, R. S. & Liang, J. Caspase family proteases and apoptosis. Acta Biochim. Biophys. Sin.37, 719–727 (2005). 10.1111/j.1745-7270.2005.00108.x [DOI] [PubMed] [Google Scholar]

[CR63] 63.Shi, Y. Mechanisms of caspase activation and inhibition during apoptosis. Mol. Cell9, 459–470 (2002). 10.1016/S1097-2765(02)00482-3 [DOI] [PubMed] [Google Scholar]

[CR64] 64.Denning, D. P., Hatch, V. & Horvitz, H. R. Both the caspase CSP-1 and a caspase-independent pathway promote programmed cell death in parallel to the canonical pathway for apoptosis in Caenorhabditis elegans. PLoS Genet.9, e1003341 (2013). 10.1371/journal.pgen.1003341 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR65] 65.Yan, W. et al. Control of PERK eIF2alpha kinase activity by the endoplasmic reticulum stress-induced molecular chaperone P58IPK. Proc. Natl Acad. Sci. USA99, 15920–15925 (2002). 10.1073/pnas.252341799 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR66] 66.Otero, J. H., Lizak, B. & Hendershot, L. M. Life and death of a BiP substrate. Semin. Cell Dev. Biol.21, 472–478 (2010). 10.1016/j.semcdb.2009.12.008 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR67] 67.Rutkowski, D. T. et al. The role of p58IPK in protecting the stressed endoplasmic reticulum. Mol. Biol. Cell18, 3681–3691 (2007). 10.1091/mbc.e07-03-0272 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR68] 68.Petrova, K., Oyadomari, S., Hendershot, L. M. & Ron, D. Regulated association of misfolded endoplasmic reticulum lumenal proteins with P58/DNAJc3. EMBO J.27, 2862–2872 (2008). 10.1038/emboj.2008.199 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR69] 69.Tao, J., Wu, Y., Ron, D. & Sha, B. Preliminary X-ray crystallographic studies of mouse UPR responsive protein P58(IPK) TPR fragment. Acta Crystallogr. Sect. F. Struct. Biol. Cryst. Commun.64, 108–110 (2008). 10.1107/S1744309108000833 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR70] 70.Munoz-Lobato, F. et al. Protective role of DNJ-27/ERdj5 in Caenorhabditis elegans models of human neurodegenerative diseases. Antioxid. Redox Signal20, 217–235 (2014). 10.1089/ars.2012.5051 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR71] 71.Wang, J., Lee, J., Liem, D. & Ping, P. HSPA5 Gene encoding Hsp70 chaperone BiP in the endoplasmic reticulum. Gene618, 14–23 (2017). 10.1016/j.gene.2017.03.005 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR72] 72.Shen, X. et al. Complementary signaling pathways regulate the unfolded protein response and are required for C. elegans development. Cell107, 893–903 (2001). 10.1016/S0092-8674(01)00612-2 [DOI] [PubMed] [Google Scholar]

[CR73] 73.Kapulkin, W. J., Hiester, B. G. & Link, C. D. Compensatory regulation among ER chaperones in C. elegans. FEBS Lett.579, 3063–3068 (2005). 10.1016/j.febslet.2005.04.062 [DOI] [PubMed] [Google Scholar]

[CR74] 74.Sriburi, R. et al. Coordinate regulation of phospholipid biosynthesis and secretory pathway gene expression in XBP-1(S)-induced endoplasmic reticulum biogenesis. J. Biol. Chem.282, 7024–7034 (2007). 10.1074/jbc.M609490200 [DOI] [PubMed] [Google Scholar]

[CR75] 75.Li, F. & Zhang, H. Lysosomal acid lipase in lipid metabolism and beyond. Arterioscler. Thromb. Vasc. Biol.39, 850–856 (2019). 10.1161/ATVBAHA.119.312136 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR76] 76.Tempio, T. et al. A virtuous cycle operated by ERp44 and ERGIC-53 guarantees proteostasis in the early secretory compartment. iScience24, 102244 (2021). 10.1016/j.isci.2021.102244 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR77] 77.Le Borgne, F., Ben Mohamed, A., Logerot, M., Garnier, E. & Demarquoy, J. Changes in carnitine octanoyltransferase activity induce alteration in fatty acid metabolism. Biochem. Biophys. Res. Commun.409, 699–704 (2011). 10.1016/j.bbrc.2011.05.068 [DOI] [PubMed] [Google Scholar]

[CR78] 78.Timson, D. J. & Reece, R. J. Identification and characterisation of human aldose 1-epimerase. FEBS Lett.543, 21–24 (2003). 10.1016/S0014-5793(03)00364-8 [DOI] [PubMed] [Google Scholar]

[CR79] 79.Jiao, X., Chang, J. H., Kilic, T., Tong, L. & Kiledjian, M. A mammalian pre-mRNA 5’ end capping quality control mechanism and an unexpected link of capping to pre-mRNA processing. Mol. Cell50, 104–115 (2013). 10.1016/j.molcel.2013.02.017 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR80] 80.Doamekpor, S. K. et al. DXO/Rai1 enzymes remove 5’-end FAD and dephospho-CoA caps on RNAs. Nucleic Acids Res.48, 6136–6148 (2020). 10.1093/nar/gkaa297 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR81] 81.Grudzien-Nogalska, E. & Kiledjian, M. New insights into decapping enzymes and selective mRNA decay. Wiley Interdiscip. Rev. RNA810.1002/wrna.1379 (2017). [DOI] [PMC free article] [PubMed]

[CR82] 82.Stelzer, G. et al. The GeneCards Suite: From Gene Data Mining to Disease Genome Sequence Analyses. Curr Protoc Bioinformatics. 54, 10.1002/cpbi.5 (2016). [DOI] [PubMed]

[CR83] 83.Sayers, E. W. et al. Database resources of the national center for biotechnology information. Nucleic Acids Res. 50, D20-D26 10.1093/nar/gkab1112 (2022). [DOI] [PMC free article] [PubMed]

[CR84] 84.Halestrap, A. P. The monocarboxylate transporter family–structure and functional characterization. IUBMB Life64, 1–9 (2012). 10.1002/iub.573 [DOI] [PubMed] [Google Scholar]

[CR85] 85.Deng, Y. et al. The Xbp1s/GalE axis links ER stress to postprandial hepatic metabolism. J. Clin. Invest.123, 455–468 (2013). 10.1172/JCI62819 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR86] 86.Huang, C., Tong, J., Bi, F., Zhou, H. & Xia, X. G. Mutant TDP-43 in motor neurons promotes the onset and progression of ALS in rats. J. Clin. Invest.122, 107–118 (2012). 10.1172/JCI59130 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR87] 87.Walker, A. K. et al. Functional recovery in new mouse models of ALS/FTLD after clearance of pathological cytoplasmic TDP-43. Acta Neuropathol.130, 643–660 (2015). 10.1007/s00401-015-1460-x [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR88] 88.Marciniak, S. J. et al. CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum. Genes Dev.18, 3066–3077 (2004). 10.1101/gad.1250704 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR89] 89.Urra, H., Dufey, E., Lisbona, F., Rojas-Rivera, D. & Hetz, C. When ER stress reaches a dead end. Biochim. Biophys. Acta1833, 3507–3517 (2013). 10.1016/j.bbamcr.2013.07.024 [DOI] [PubMed] [Google Scholar]

[CR90] 90.Imanikia, S., Ozbey, N. P., Krueger, C., Casanueva, M. O. & Taylor, R. C. Neuronal XBP-1 activates intestinal lysosomes to improve proteostasis in C. elegans. Curr. Biol.29, 2322–2338.e2327 (2019). 10.1016/j.cub.2019.06.031 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR91] 91.Zarouchlioti, C., Parfitt, D. A., Li, W., Gittings, L. M. & Cheetham, M. E. DNAJ proteins in neurodegeneration: essential and protective factors. Philos. Trans. R. Soc. Lond. B Biol. Sci.37310.1098/rstb.2016.0534 (2018). [DOI] [PMC free article] [PubMed]

[CR92] 92.Damiano, F., Testini, M., Tocci, R., Gnoni, G. V. & Siculella, L. Translational control of human acetyl-CoA carboxylase 1 mRNA is mediated by an internal ribosome entry site in response to ER stress, serum deprivation or hypoxia mimetic CoCl(2). Biochim. Biophys. Acta Mol. Cell Biol. Lipids1863, 388–398 (2018). 10.1016/j.bbalip.2018.01.006 [DOI] [PubMed] [Google Scholar]

[CR93] 93.Morrison, J. K., Friday, A. J., Henderson, M. A., Hao, E. & Keiper, B. D. Induction of cap-independent BiP (hsp-3) and Bcl-2 (ced-9) translation in response to eIF4G (IFG-1) depletion in C. elegans. Translation2, e28935 (2014). 10.4161/trla.28935 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR94] 94.Imanikia, S., Sheng, M., Castro, C., Griffin, J. L. & Taylor, R. C. XBP-1 remodels lipid metabolism to extend longevity. Cell Rep.28, 581–589.e584 (2019). 10.1016/j.celrep.2019.06.057 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR95] 95.Daniele, J. R. et al. UPR(ER) promotes lipophagy independent of chaperones to extend life span. Sci. Adv.6, eaaz1441 (2020). 10.1126/sciadv.aaz1441 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR96] 96.Ho, N. et al. Stress sensor Ire1 deploys a divergent transcriptional program in response to lipid bilayer stress. J. Cell Biol.21910.1083/jcb.201909165 (2020). [DOI] [PMC free article] [PubMed]

[CR97] 97.Piperi, C., Adamopoulos, C. & Papavassiliou, A. G. XBP1: a pivotal transcriptional regulator of glucose and lipid metabolism. Trends Endocrinol. Metab.27, 119–122 (2016). 10.1016/j.tem.2016.01.001 [DOI] [PubMed] [Google Scholar]

[CR98] 98.Ozbey, N. P. et al. Tyramine acts downstream of neuronal XBP-1s to coordinate inter-tissue UPR(ER) activation and behavior in C. elegans. Dev. Cell55, 754–770.e756 (2020). 10.1016/j.devcel.2020.10.024 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR99] 99.Ellis, H. M. & Horvitz, H. R. Genetic control of programmed cell death in the nematode C. elegans. Cell44, 817–829 (1986). 10.1016/0092-8674(86)90004-8 [DOI] [PubMed] [Google Scholar]

[CR100] 100.Shaham, S. Identification of multiple Caenorhabditis elegans caspases and their potential roles in proteolytic cascades. J. Biol. Chem.273, 35109–35117 (1998). 10.1074/jbc.273.52.35109 [DOI] [PubMed] [Google Scholar]

[CR101] 101.Slee, E. A., Adrain, C. & Martin, S. J. Executioner caspase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis. J. Biol. Chem.276, 7320–7326 (2001). 10.1074/jbc.M008363200 [DOI] [PubMed] [Google Scholar]

[CR102] 102.Hitomi, J. et al. Apoptosis induced by endoplasmic reticulum stress depends on activation of caspase-3 via caspase-12. Neurosci. Lett.357, 127–130 (2004). 10.1016/j.neulet.2003.12.080 [DOI] [PubMed] [Google Scholar]

[CR103] 103.Kaufman, R. J. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev.13, 1211–1233 (1999). 10.1101/gad.13.10.1211 [DOI] [PubMed] [Google Scholar]

[CR104] 104.Guo, H. et al. Active caspase-6 and caspase-6-cleaved tau in neuropil threads, neuritic plaques, and neurofibrillary tangles of Alzheimer’s disease. Am. J. Pathol.165, 523–531 (2004). 10.1016/S0002-9440(10)63317-2 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR105] 105.Selznick, L. A. et al. In situ immunodetection of neuronal caspase-3 activation in Alzheimer disease. J. Neuropathol. Exp. Neurol.58, 1020–1026 (1999). 10.1097/00005072-199909000-00012 [DOI] [PubMed] [Google Scholar]

[CR106] 106.Wai, M. S. et al. Co-localization of hyperphosphorylated tau and caspases in the brainstem of Alzheimer’s disease patients. Biogerontology10, 457–469 (2009). 10.1007/s10522-008-9189-8 [DOI] [PubMed] [Google Scholar]

[CR107] 107.Wang, Z. et al. Sustained overexpression of spliced X-box-binding protein-1 in neurons leads to spontaneous seizures and sudden death in mice. Commun. Biol.6, 252 (2023). 10.1038/s42003-023-04594-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR108] 108.Martinez, G. et al. Regulation of memory formation by the transcription factor XBP1. Cell Rep.14, 1382–1394 (2016). 10.1016/j.celrep.2016.01.028 [DOI] [PubMed] [Google Scholar]

[CR109] 109.Duran-Aniotz, C. et al. The unfolded protein response transcription factor XBP1s ameliorates Alzheimer’s disease by improving synaptic function and proteostasis. Mol. Ther.31, 2240–2256 (2023). 10.1016/j.ymthe.2023.03.028 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR110] 110.Cisse, M. et al. The transcription factor XBP1s restores hippocampal synaptic plasticity and memory by control of the Kalirin-7 pathway in Alzheimer model. Mol. Psychiatry22, 1562–1575 (2017). 10.1038/mp.2016.152 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR111] 111.Chatterjee, S. et al. Endoplasmic reticulum chaperone genes encode effectors of long-term memory. Sci. Adv.8, eabm6063 (2022). 10.1126/sciadv.abm6063 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR112] 112.Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods6, 343–345 (2009). 10.1038/nmeth.1318 [DOI] [PubMed] [Google Scholar]

[CR113] 113.Brenner, S. The genetics of Caenorhabditis elegans. Genetics77, 71–94 (1974). 10.1093/genetics/77.1.71 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR114] 114.American Society for Cell Biology, Epstein, H. F. & Shakes, D. C. in Methods in Cell Biology, 1 online resource (xxi) 659 (Academic Press, 1995).

[CR115] 115.Wood, W. B. The Nematode Caenorhabditis elegans (Cold Spring Harbor Laboratory, 1988).

[CR116] 116.Paix, A., Folkmann, A., Rasoloson, D. & Seydoux, G. High efficiency, homology-directed genome editing in Caenorhabditis elegans using CRISPR-Cas9 ribonucleoprotein complexes. Genetics201, 47–54 (2015). 10.1534/genetics.115.179382 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR117] 117.Currey, H. N. & Liachko, N. F. Evaluation of motor impairment in C. elegans models of amyotrophic lateral sclerosis. J. Vis. Exp.10.3791/62699 (2021). [DOI] [PubMed]

[CR118] 118.Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH image to ImageJ: 25 years of image analysis. Nat. Methods9, 671–675 (2012). 10.1038/nmeth.2089 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR119] 119.Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol.215, 403–410 (1990). 10.1016/S0022-2836(05)80360-2 [DOI] [PubMed] [Google Scholar]

[CR120] 120.Sherman, B. T. et al. DAVID: a web server for functional enrichment analysis and functional annotation of gene lists (2021 update). Nucleic Acids Res.50, W216–W221 (2022). 10.1093/nar/gkac194 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR121] 121.Tang, D. et al. SRplot: a free online platform for data visualization and graphing. PLoS ONE18, e0294236 (2023). 10.1371/journal.pone.0294236 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Endoplasmic reticulum unfolded protein response transcriptional targets of XBP-1s mediate rescue from tauopathy

Sarah M Waldherr

Marina Han

Aleen D Saxton

Taylor A Vadset

Pamela J McMillan

Jeanna M Wheeler

Nicole F Liachko

Brian C Kraemer

Abstract

Introduction

Fig. 1. Identification of tauopathy-related XBP-1s/ATF6 target genes.

Results

Transcriptomic analysis reveals candidate XBP-1s target genes involved in tauopathy suppression in C. elegans

Table 1.

Table 2.

A suite of ATF6-dependent XBP-1s target genes is required for C. elegans tauopathy suppression

Fig. 2. Cytoplasmic resident caspase upregulation by XBP-1s/ATF6 is required for suppression of tauopathy.

Fig. 3. ER resident co-chaperone upregulation by XBP-1s/ATF6 is required for suppression of tauopathy.

Fig. 4. ER resident chaperone HSP-4/BiP upregulation by XBP-1s/ATF6 is required for suppression of tauopathy.

Fig. 5. Cytoplasmic resident choline kinase CKB-2 and lysosomal resident lipase LIPL-3 upregulation by XBP-1s/ATF6 is required for suppression of tauopathy.

A suite of other XBP-1s target genes is also required for C. elegans tauopathy suppression

Fig. 6. Additional XBP-1s target genes are required for suppression of tauopathy.

XBP1s induction upregulates tauopathy-related target genes, but causes lethality in mice

Fig. 7. niXBP1s activation in mouse brain upregulates tauopathy-related target genes.

Discussion

Fig. 8. XBP-1s transcriptional remodeling requires concerted upregulation of a suite of genes for tauopathy suppression.

Methods

Plasmids

C. elegans strains and transgenics

C. elegans behavioral analysis

Manual swimming assay

Automated swimming assay

Radial locomotion assay

C. elegans protein extraction

Protein immunoblotting

C. elegans RNA purification

Library construction and transcriptomic analysis

Work with mice

Immunohistochemical evaluation

Statistics and reproducibility

Schematic illustrations

Reporting summary

Supplementary information

Acknowledgements

Author contributions

Peer review

Peer review information

Data availability

Competing interests

Footnotes

Supplementary information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases