Human tau compromises neuronal structural integrity in C. elegans promoted by age, stress and phosphorylation

Human tau compromises neuronal structural integrity in C. elegans promoted by age, stress and phosphorylation | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Human tau compromises neuronal structural integrity in C. elegans promoted by age, stress and phosphorylation View ORCID Profile Maithili Joshi , View ORCID Profile Sanne van Falier , View ORCID Profile Tessa Sinnige doi: https://doi.org/10.1101/2025.08.01.668095 Maithili Joshi 1 Molecular Biophysics & Biochemistry, Bijvoet Centre for Biomolecular Research, Utrecht University , Padualaan 8, 3584 CH Utrecht, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Maithili Joshi Sanne van Falier 1 Molecular Biophysics & Biochemistry, Bijvoet Centre for Biomolecular Research, Utrecht University , Padualaan 8, 3584 CH Utrecht, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sanne van Falier Tessa Sinnige 1 Molecular Biophysics & Biochemistry, Bijvoet Centre for Biomolecular Research, Utrecht University , Padualaan 8, 3584 CH Utrecht, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tessa Sinnige For correspondence: t.sinnige1{at}uu.nl Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Tauopathies are a group of progressive neurodegenerative diseases amongst which Alzheimer’s disease is the most prevalent. They typically have a late age of onset and lead to cognitive decline. Tau is a highly soluble, intrinsically disordered microtubule binding protein that aggregates in these diseases. The mechanisms by which age-associated changes alter tau functionality and compromise neuronal structural integrity remain poorly understood. Here, we show with the use of single-copy gene insertion Caenorhabditis elegans models that human tau preferentially localises to neuronal processes in the nematode, consistent with its function of binding to microtubules. The expression of tau in the nervous system leads to age-associated changes in neuronal structural integrity. The observed “buckling” phenotype was exacerbated by exposure of the animals to different stressors which are known to enhance tau phosphorylation. Expressing phosphomimic tau or co-expressing human kinases both severely worsened the buckling phenotype. Kinase expression additionally induced tau inclusions in a small proportion of animals. These results point to an interplay between ageing, stress and phosphorylation leading to structural changes in neuronal processes prior to widespread tau aggregation. Introduction Tauopathies are a heterogenous group of neurodegenerative diseases characterised by the accumulation of the protein tau in the form of neurofibrillary tangles ( Creekmore et al., 2024 ; Götz et al., 2019 ; Wang & Mandelkow, 2016 ). Primary tauopathies such as frontotemporal dementia (FTD) are driven by mutations in tau, and display neuronal and glial tau tangles as the main pathological hallmark. Alzheimer’s disease (AD) is the most prevalent secondary tauopathy, featuring extracellular amyloid plaques next to intraneuronal tau tangles. AD is largely sporadic, with age being the main risk factor next to genetic background and lifestyle ( Scheltens et al., 2021 ) Tau is an intrinsically disordered protein known to bind to microtubules. It is produced as six different isoforms, varying from 352 to 441 amino acids in length. These isoforms carry either 0, 1 or 2 N-terminal inserts combined with either 3 or 4 tandem repeats of an amino acid motif which is the attachment site to microtubules, known as the microtubule binding repeat region (MTBR) ( Götz et al., 2019 ). In humans, tau is distributed throughout the neuron in early development, whereas in mature neurons it is localised to axons. Tau pathology is accompanied by mislocalisation to the cell body, where neurofibrillary tangles are found ( Götz et al., 2019 ). Post-translational modifications are known to play a key role in this pathological process, most notably phosphorylation ( Alquezar et al., 2021 ; Basheer et al., 2023 ; Wegmann et al., 2021 ). It is hypothesized that hyperphosphorylation of tau causes the protein to detach from microtubules, leading to an increased concentration of free cytosolic tau. This can lead to aggregation of tau and neurotoxicity ( Wang & Mandelkow, 2016 ; Wegmann et al., 2021 ). However, the sequence of these events and the exact cause of toxicity remain unclear. The tau protein and tau-associated pathologies are not unique to humans ( Edler et al., 2017 ; Oikawa et al., 2010 ). Microtubule-associated proteins (MAPs) of the tau/MAP2/MAP4 family are found throughout much of the animal kingdom ( Dehmelt & Halpain, 2005 ). The nematode Caenorhabditis elegans has one homologue of tau/MAP like proteins known as PTL-1 (protein with tau like repeats-1), which localises to both axons and dendrites and provides structural integrity to the neurons ( Goedert et al., 1996 ). It is mostly expressed in the mechanosensory touch receptor neurons, which contain unusual 15 protofilament microtubules, in larval and adult stages ( Goedert et al., 1996 ; Taylor et al., 2021 ). C. elegans has been employed as a model organism to study both functional and pathological aspects of PTL-1 and human tau. PTL-1 was found to be important for maintaining neuronal health during ageing, and either increasing or decreasing its levels leads to loss of structural integrity of the touch receptor neurons ( Chew et al., 2013 ). PTL-1 is involved in retrograde transport ( Tien et al., 2011 ), and is involved in mechanosensation but without visibly modulating microtubule organisation ( Gordon et al., 2008 ). Studies aiming to understand human tau pathology have been mostly based on neuronal overexpression of different wild type and mutant tau constructs. These typically reported functional defects accompanied by damage to neuronal structures ( Brandt et al., 2009 ; Fatouros et al., 2012 ; Kraemer et al., 2003 ; Miyasaka et al., 2005 ; Pir et al., 2016 ) In some cases, insoluble tau was found to accumulate ( Aquino Nunez et al., 2022 ; Fatouros et al., 2012 ; Kraemer et al., 2003 ), suggesting that tau aggregation is linked to the observed phenotypes. In a different approach, wild type tau was expressed from a single-copy gene insertion in the touch receptor neurons, which did not lead to functional or morphological impairments. However, mutations mimicking certain post-translational modifications affected neuronal structure and function, while causing mitochondrial fragmentation ( Guha et al., 2020 ). In another recent study, panneuronal single-copy tau strains also lacked observable phenotypes, whereas multicopy strains displayed motility impairments correlating with expression levels ( Han et al., 2024 ). Despite these advances, the mechanisms by which disease-associated changes alter tau functionality and compromise neuronal structural integrity remain poorly understood. Here, we created single-copy gene insertion C. elegans models expressing human wild type and mutant tau in the nervous system and found an age-associated phenotype, in which the ventral nerve cord displayed structural abnormalities. This phenotype was worsened by cellular stress and phosphorylation prior to the formation of observable aggregates, thus shedding light on the early pathological events associated with human tau expression. Results Human tau is enriched in neuronal processes in C. elegans C. elegans single-copy strains were made based on the pan-neuronal expression of wild type and P301L mutant tau using the rgef-1 promotor region. P301L is a disease associated mutation involved in frontotemporal dementia. For both strains, the 383 amino acid long 0N4R isoform of tau was used ( Fig.1A ), N-terminally tagged with monomeric enhanced yellow fluorescent protein (mEYFP). A single-copy strain expressing mEYFP with the same promotor served as the control. Download figure Open in new tab Fig. 1 Human tau preferentially localises to neuronal processes in C. elegans . (A) Schematic design for tau0N4R, amino acids are numbered according to the longest tau isoform (2N4R). (B, C, D) Confocal images of the head region of mEYFP (B), tau (C) and tau P301L (D) C. elegans strains on day 1 of adulthood. Arrows show nerve ring; arrow heads the ventral nerve cord. Scale bars are 20 μm. (E) Quantification of the ratio of fluorescence intensities in neurites/cell bodies in mEYFP, tau and tau P301L strains on day 1 of adulthood (n = 8 animals per strain). Error bars are standard deviation. A Welch ANOVA was employed. (F) Quantification of the motility assay. n = 40 animals per condition. Error bars are standard deviation. (For E and F, *p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001, ns not significant.) (G) Survival assay. Dashed line indicates the median lifespan. n = 100 animals for each strain. Confocal imaging revealed that the protein localisation of wild type and mutant tau differs from that of mEYFP ( Fig.1B, C, D ). The fluorescence intensity is uniformly distributed across cell bodies and neurites in mEYFP. Contrastingly, wild type and mutant tau appear depleted in cell bodies and show a strong intensity in the nerve ring and ventral nerve cord ( Fig. 1C, D arrows and arrow heads, respectively). Quantification of the ratio of fluorescence intensity in neurites versus cell bodies was done for the three strains. The ratio for mEYFP was close to 1, in agreement with a homogeneous distribution of the protein. For both wild type and mutant tau this ratio was around 1.7, suggesting that tau is enriched in the axons and dendrites compared to neuronal cell bodies ( Fig. 1E ). This preferential localisation of tau to neuronal processes is consistent with its function of binding to microtubules. To assess the health of the animals upon the expression of tau, motility and survival assays were performed. As expected, we observed a reduction in motility at old age, but there were no significant differences between the three strains ( Fig. 1F ). Next, a survival assay was performed. The median lifespan was 16 days for mEYFP and tau, and 15 days for tauP301L ( Fig. 1G ). The restricted mean survival time (RMST), corresponding to the area under the survival curve, was 17 days for mEYFP and tau and 15 days for tauP301L, suggesting that the mutation has a mild effect on lifespan. Tau expression causes buckling of the ventral nerve cord with age To investigate age-related changes in the distribution of wild type and mutant tau, time course confocal imaging was done from day 1 to day 12 of adulthood. We found that some aged animals displayed a wavy appearance of the ventral nerve cord, which is a bundle of processes derived from interneurons and motor neurons ( Fig. 2A ). The wavy appearance has been previously reported to occur in ageing touch receptor neurons ( Pan et al., 2011 ; Toth et al., 2012 ), and it has been linked to defects in β-spectrin ( Krieg et al., 2014 ) as well as the microtubule cytoskeleton ( Hsu et al., 2014 ; Krieg et al., 2017 ; Topalidou et al., 2012 ). This phenotype is also referred to as buckling, which is the term that we will use from here on. Download figure Open in new tab Fig. 2 Tau expression induces buckling of the ventral nerve cord with age. (A) Representative confocal images of the head region of mEYFP, tau and tau P301L strains from day 1 to day 12 of adulthood. Buckling of the ventral nerve cord is indicated by red arrows. Scale bars are 20 μm. (B) Quantification of the percentage of animals showing buckling of the ventral nerve cord. n = 20 animals per condition for each of 2 biological repeats. Error bars are standard deviation. A two-way ANOVA was employed (*p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001). The percentage of animals displaying buckling of the ventral nerve cord increases with age, and only becomes notable for the mEYFP control of day 12 ( Fig. 2B ). However, the presence of tau exasperates this phenotype, appearing at earlier timepoints and in a larger fraction of animals. Wild type and P301L tau show a comparable percentage of buckling animals at all timepoints, implying that the mutation does not worsen neuronal structural integrity ( Fig. 2B ). We did not observe other changes in protein distribution, such as macroscopically visible protein inclusions. Buckling phenotype is induced at younger age by cellular stress Next, we examined if the buckling phenotype is affected by environmental stressors. C. elegans strains expressing mEYFP, tau and tau P301L were exposed to different stressors on day 4 of adulthood and inspected for neuronal buckling on the next day ( Fig. 3A ). Consistent with the previous results, very few animals show the buckling phenotype on day 5 of adulthood in the absence of stress ( Fig. 3B and C ). Download figure Open in new tab Fig. 3 Different types of stress exacerbate the buckling phenotype of tau animals. (A) Schematic showing the experimental set-up. (B) Quantification of the percentage of animals showing buckling of ventral nerve cord exposed to heat and UV. (C) Quantification of neuronal buckling upon sodium arsenite and DTT treatment. n = 20 animals per condition for each of 3 biological repeats. Error bars are standard deviation. A two-way ANOVA was employed (*p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001). We tested different types of stress, associated with different cellular events. UV radiation causes an increase in reactive oxygen species in the cell leading to DNA damage, which has been linked to tau pathology ( Asada-Utsugi et al., 2022 ). Upon exposure to UV radiation, the buckling phenotype increased to around 35 - 40 % of animals expressing wild type and mutant tau, whereas those expressing mEYFP were affected to a much smaller extent ( Fig. 3B ). Contrastingly, a 2 h heat shock, which promotes protein misfolding and leads to induction of the heat shock response, had no effect on the buckling phenotype ( Fig. 3C ). Sodium arsenite is a form of heavy metal stress that has been linked to tau phosphorylation and neurodegeneration ( Giasson et al., 2002 ; Wisessaowapak et al., 2022 ). Exposure to sodium arsenite increases the buckling phenotype to 20 - 25 % of animals expressing wild type and mutant tau ( Fig. 3C ). Dithiothreitol (DTT) compromises disulfide bridge formation and causes endoplasmic reticulum (ER) stress ( Gokul & Singh, 2022 ). Exposure to DTT caused buckling in 25 - 30 % of wild type and mutant tau expressing animals. Similarly as in the ageing experiment ( Fig. 2 ), we did not observe any significant differences between wild type and mutant tau for the stressors that had an effect. As most of the processes in the ventral nerve cord are from motor neurons, we checked if exposure to stress causes a decline in motility. The animals were treated in the same way as before, exposing age-synchronised day 4 adults to stress and carrying out the motility assay on day 5, but no effect was seen on their motility (Fig. S1). UV treated animals show lower motility compared to control and other treatments, but there is no difference between mEYFP and tau expressing worms (Fig. S1). Thus, although there is impact on neuronal structure in animals expressing tau exposed to stress, we could not detect a reduction in their functionality. Buckling phenotype is exacerbated by phosphomimic substitutions Tau hyperphosphorylation has been linked to its aggregation and the associated neurodegeneration. To understand the impact of tau phosphorylation on the nervous system of C. elegans , we created a single-copy model pan-neuronally expressing phosphomimic tau E14. This construct has 14 disease-associated serine/threonine phosphorylation sites mutated to glutamate to mimic phosphorylation ( Fig. 4A ), and has been reported to enhance toxicity in the brain of Drosophila melanogaster ( Dias-Santagata et al., 2007 ; Khurana et al., 2006 ) and neuronal cell culture ( Hallinan et al., 2019 ; Padmanabhan et al., 2024 ). Download figure Open in new tab Fig. 4 Expression of phosphomimic tau exacerbates tau-induced neuronal buckling. (A) Schematic showing the positions of the phosphomimic substitutions on the tau 0N4R construct. Amino acids are numbered according to the longest tau isoform (2N4R). (B) Quantification of the percentage of animals showing buckling of ventral nerve cord for tau, tau P301L and tau E14 strains. n = 20 animals per condition for each of 2 biological repeats. Data shown are average ± standard deviation. A two-way ANOVA was employed (*p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001). (C) Quantification of the survival assay. n = 100 animals for each strain. Expression of tau E14 led to a higher percentage of animals showing neuronal buckling compared to both wild type and mutant tau, reaching statistical significance from day 8 of adulthood onwards ( Fig. 4B ). Thus, the phosphomimic substitutions have an impact on neuronal structural integrity in C. elegans . A survival assay was also done to assess the health of these animals. Tau E14 had a median lifespan of 15 days compared to 16 days for mEYFP ( Fig. 4C ). The RMST for tau E14 was 16 days compared to 17 days for mEYFP, indicating a mild effect of the phosphomimic mutations on the animals’ survival. Overexpression of human kinases enhances tau-associated phenotypes Since phosphomimic substitutions have a different charge (−1) compared to phosphorylation (−2), they may impact the properties of the protein differentially. Hence, to also assess the effect of direct phosphorylation, we overexpressed the human kinases GSK-3β and CDK5 pan-neuronally under control of the rab-3 promotor in wild type animals. These animals were subsequently crossed with mEYFP, tau and tau P301L strains. GSK-3β and CDK5 are both serine/threonine kinases that have been implicated in Alzheimer’s disease. CDK5 is reported to enhance both tau accumulation and neurodegeneration in Drosophila and mouse models ( Noble et al., 2003 ; Saito et al., 2019 ). GSK-3β is involved in many cellular pathways and is another key kinase in tau hyperphosphorylation and neurodegeneration ( Lauretti et al., 2020 ). Expression of both GSK-3β and CDK5 in animals expressing wild type tau caused earlier onset and a higher percentage of animals showing neuronal buckling ( Fig. 5A ). A similar effect was seen for tau P301L, where the increase in buckling was significant for both kinases on days 5 and 12 ( Fig. 5B ). The phenotype of controls expressing mEYFP was considerably less affected by the kinases (Fig. S2A). The data thus suggest that the expression of both kinases enhances buckling through increased tau phosphorylation. Download figure Open in new tab Fig. 5 Expression of human kinases enhances tau-associated neuronal buckling. (A) Quantification of the percentage of animals showing buckling of ventral nerve cord with age for tau, tau x GSK-3β and tau x CDK5. (B) Quantification of the percentage of animals showing buckling of ventral nerve cord with age for tau P301L, tau P301L x GSK-3β and tau P301L x CDK5. n = 20 animals per condition for each of 2 biological repeats. Data shown are average ± standard deviation. A two-way ANOVA was employed (*p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001). (C) Confocal images of tau animals crossed with GSK-3β and CDK5 on day 10 (top row) and day 16 (bottom row) of adulthood. Inclusions are marked with red arrows. Scale bars are 20 μm. Insets show close-ups of the regions with inclusions; scale bars are 5 μm. Confocal imaging of the strains expressing kinases on day 10 and day 16 of adulthood furthermore showed that some animals had formed tau inclusions, which appear as bright spots and are mostly localised to the posterior of the nerve ring rather than the ventral nerve cord ( Fig. 5C ). This was a rare occurrence with very few of the kinase expressing animals showing inclusions, yet none of the control animals displayed this feature as late as day 16 (Fig. S2B and C). In some images, we could discriminate that the inclusion appeared to be localised to the cell body in a perinuclear region (asterisks in Fig. 5C ). This observation strikingly resembles the formation of tau tangles in neuronal cell bodies, away from the physiological microtubule binding sites, that is observed in human Alzheimer’s disease. Discussion In this study, C. elegans single-copy models pan-neuronally expressing human tau protein tagged with mEYFP were created. Using these models, we discovered that human tau compromises the structural integrity of the nervous system under conditions known to play a role in tauopathies, including age, cellular stress and phosphorylation. Although the general health of the animals is largely unaffected as judged from motility and survival assays, time course confocal imaging revealed that animals expressing tau show wavy neuronal processes in the ventral nerve cord as they age. The phenotype is first observed between day 5 and 8 of adulthood, which is largely post-reproduction, and increases in frequency as the animals age further ( Fig. 2 ). The buckling phenotype has been observed before as a consequence of ageing ( Pan et al., 2011 ; Toth et al., 2012 ) or aberrations in both the actin/β-spectrin ( Krieg et al., 2014 ) and the microtubule cytoskeleton ( Hsu et al., 2014 ; Krieg et al., 2017 ; Topalidou et al., 2012 ). These studies have mostly focused on the six touch receptor neurons (ALML, ALMR, PLML, PLMR, AVM and PVM), in which the C. elegans tau homolog PTL-1 is natively expressed. Deletion of PTL-1 was shown to lead to structural defects in these neurons, such as blebbing and branching ( Chew et al., 2013 ). Interestingly, expressing additional copies of either PTL-1 or human tau in wild type background caused similar defects, suggesting that either loss-of-function or gain-of-function lead to the same phenotype ( Chew et al., 2013 ). Here, we extend these observations by showing that pan-neuronally expressed human tau induces structural defects in the much larger subset of neurons that make up the ventral nerve cord. This defect seems to be related to tau pathology rather than its physiological function, given that it only appears in aged animals and is worsened by cellular stress and phosphorylation. UV radiation, heavy metal stress and ER stress induced the buckling phenotype at earlier onset, showing a considerable number of affected animals on day 5 of adulthood ( Fig. 3 ). While these stressors can impact cellular homeostasis in multiple ways, they have each been linked to tau phosphorylation. Exposure to UV radiation causes an increase in tau phosphorylation ( Asada-Utsugi et al., 2022 ) and has been linked to activation of GSK-3β ( Lee et al., 2007 ). Similarly, induction of the unfolded protein response (UPR) through ER stress is associated with tau phosphorylation ( van der Harg et al., 2014 ) through the activity of GSK-3β ( Nijholt et al., 2013 ; Song et al., 2002 ). Finally, heavy metal stress from arsenite has also been reported to cause tau hyperphosphorylation ( Giasson et al., 2002 ; Wisessaowapak et al., 2022 ). Our model thus appears to recapitulate these cellular pathways, showing that they result in the loss of neuronal structural integrity. To explore further how tau phosphorylation affects neuronal structure, we created a strain expressing a phosphomimic version of tau (E14) and saw that a higher percentage of animals showed buckling of the ventral nerve cord ( Fig. 4B ). This was also the case when two kinases, GSK-3β and CDK5, were overexpressed in the context of the tau single copy lines ( Fig. 5A, B ). The majority of the tau signal was still present in the neuronal processes, suggesting that the phosphorylated tau species remain largely associated with microtubules. However, kinase expression induced the formation of tau inclusions in some animals at very old age, starting from day 10 of adulthood ( Fig. 5C ). Thus, it appears that trafficking and accumulation of tau in the cell bodies occur at a later stage in our model than its phosphorylation and the associated structural deficits of the neuronal processes. These findings could be explained by a mechanism wherein tau undergoes phosphorylation in the neuronal processes, leading to aberrant conformations that interfere with functional microtubule dynamics. It is interesting in this regard that tau has been suggested to form functional oligomers or condensates on microtubule surfaces ( Gyparaki et al., 2021 ; Hernández-Vega et al., 2017 ; Tan et al., 2019 ). However, these assemblies can transition into pathological aggregate species and ultimately the fibril structures that are found in neurofibrillary tangles in AD ( Tan et al., 2019 ; Wegmann et al., 2018 ). We propose that small multimeric tau species are responsible for the structural defects in the nerve cord, and that these eventually dissociate from the microtubules and get trafficked to cell bodies where further accumulation and aggregation takes place. It is somewhat surprising that the phenotype of the disease-associated P301L mutant is not worse than that of wild type tau in our C. elegans models, other than a subtle reduction in lifespan. Tau P301L is known to bind to microtubules with reduced affinity, and slows down microtubule assembly ( Hong, 1998 ; Rizzu et al., 1999 ). Its aggregation propensity is also higher, which has been suggested to be due to local unfolding close to the amyloid motif ( Chen et al., 2019 ). It is possible that the effects of the P301L mutation only occur at the late stages of tau mislocalisation and accumulation in the cell bodies, but the lifespan of the animals precluded investigations at later timepoints beyond day 16. Altogether, our study shows that C. elegans neurons undergo tau-associated structural changes with age, which are enhanced by cellular stress and tau phosphorylation. The data suggest that the loss of neuronal structural integrity precedes the formation of tau inclusions. The C. elegans models here presented may thus provide new opportunities to study the early stages of tau pathology, and look for modifiers that restore these. Materials and methods Molecular biology Plasmids used in the study were generated using Gibson assembly (NEB) and are listed in Table S1. The pCFJ150 vector was used as backbone. For pan-neuronal expression, the promotor region of rgef-1 was used. The promotor and 3’-UTR of let-858 were amplified from C. elegans genomic DNA. EYFP was amplified from pPD30.38, which was a gift from Andrew Fire (Addgene plasmid # 1443; http://n2t.net/addgene:1443 ; RRID:Addgene_1443) and mutated (A206K) by site directed mutagenesis (Q5 site-directed mutagenesis kit, NEB) to create mEYFP. Tau was ordered as synthetic DNA from IDT. Site-directed mutagenesis was used to make TauP301L (Phusion Site-Directed Mutagenesis Kit, Thermo Fisher Scientific). Tau E14, GSK-3β and CDK5 were ordered as synthetic DNA from Thermo Fisher Scientific. All oligonucleotides used in this study were ordered from IDT and are listed in Table S2. C. elegans strain generation C. elegans strains used in this study are listed in Table S3. Single-copy strains were generated using the MosSCI method ( Frøkjær-Jensen et al., 2008 ). An injection mix containing 10 ng/µL plasmid of interest, 50 ng/µL pCFJ601 (transposase), 10 ng/µL pMA122 (negative selection marker) and 30 ng/µL ccGFP (coelomocyte co-injection marker) was injected into the gonads of EG6699 adults. These were incubated at 25 °C for one week followed by a 2 h heat shock at 34 °C. The plates were chunked and screened for fluorescent animals with rescued movement and lacking expression of the co-injection maker. The insertion of the transgene was confirmed by amplifying the insert using PCR with LongAmp Taq DNA polymerase (NEB) followed by sequencing. The animals were backcrossed with N2 five times to dilute mutations induced by the heat shock. For the kinases, a mixture of 15 ng/µL of the plasmid of interest, 15 ng/µL of ccRFP and 70 ng/ µL 1 kb DNA ladder (NEB) was injected into N2 animals. The extra-chromosomal lines were then crossed with mEYFP, tau and tau P301L single copy lines. C. elegans strain maintenance All strains were maintained on nematode growth media (NGM) seeded with E. coli OP50 at 15 °C. Age-synchronisation for time-course experiments was done by allowing adult animals to lay eggs for 2 h at 20 °C, followed by incubation at 20 °C for 3 days to reach day 1 of adulthood. Adult animals were transferred daily to fresh NGM plates to separate them from their offspring until they ceased to lay eggs. Microscopy Leica MZ10F and Leica M165FC stereomicroscopes were used for the screening and selection of transgenic animals with filter set ET YFP with excitation 500/20 nm and emission 535/30 nm. Samples for confocal imaging or for scoring of neuronal buckling phenotype were prepared by immobilising adult animals on 2.5 % agarose pads in a drop of 10 mM tetramisole in M9 (22 mM KH 2 PO 4 , 42 mM Na 2 HPO 4 , 8.5 mM NaCl, 18.7 mM NH 4 Cl, 1 mM MgSO 4 ). Confocal microscopy was done on a Nikon Eclipse Ti microscope body equipped with CSU-X1-A1 spinning disk (Yokogawa) and Nikon Plan Apo VC 60x /1.40 oil objective. The excitation and emissions wavelengths used for mEYFP were 488 nm and 525/50 nm respectively. The laser intensity used was 30 % and exposure time was 250 ms. To score animals for the buckling phenotype, a Leica DMi8 microscope with a Leica HC PL fluotar 40x oil objective with excitation wavelength 500/20 nm and emission wavelength 535/30 nm was used. Motility assays For motility assays, 5-6 age synchronised animals were transferred to a drop of 75 µL M9 buffer in a 12 well plate and recorded for 30 s on a Leica S9i microscope at 26 fps. Animals were monitored until day 16 for the time course experiments, and on day 5 for the stress experiments (24 h post exposure to stress). A total of 40 animals per condition were analysed. Stress exposure For exposing the animals to different stressors, an age-synchronous population was grown until day 4 of adulthood. For heat stress, animals were put at 34 °C for 2 h on seeded NGM plates. For UV stress, animals were transferred to unseeded plates and then irradiated with 30 mJ/cm 2 UV light using a UVP Crosslinker CL-3000 (Analytik Jena). For sodium arsenite exposure, animals were incubated for 2 h in a 5 mM solution of sodium arsenite prepared in M9 buffer. For DTT exposure, animals were incubated for 2 h in a 3 mM solution of DTT prepared in M9 buffer. As a control for these experiments, animals were incubated in M9 for 2 h. Survival assays 100 age-synchronised animals were picked for each strain on day 1 of adulthood. Animals were inspected daily and scored as dead if they were unresponsive to touch. This was done until all the animals were dead. Animals that were lost due to burrowing under the agar were censored and taken out of the analysis. Data analysis All images and videos for motility assay were analysed using imageJ. The wrMTrck plugin in imageJ ( Nussbaum-Krammer et al., 2015 ) was used to quantify body bends per second for motility assays. For statistical analysis of motility assays and neuronal buckling, a two-way ANOVA was performed in GraphPad Prism 10. For statistical analysis of the comparison of the neurite/cell bodies ratios, a Brown-Forsythe and Welch ANOVA test was performed. All data are presented as mean values with standard deviation as error bars. Standard p values (*p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001) were used for determining significance. Analysis of lifespan was done using the Kaplan-Meier method. Competing interests No competing interests declared. Funding This work was funded by a start-up grant from Utrecht University to T.S. Data and resource availability All relevant data and details of resources can be found within the article and its supplementary information. Author contributions statement Conceptualisation: M.J., T.S., Methodology: M.J., S.v.F., Formal analysis: M.J., S.v.F., T.S., Investigation: M.J., S.v.F., Writing – original draft preparation: M.J., Writing – review and editing: M.J., S.v.F, T.S., Visualisation: M.J., Supervision: T.S. Acknowledgements We thank the Biology Imaging Centre at Utrecht University for use of the confocal spinning disk microscope. We are grateful to Jorieke Tiggelaar for help with strain generation and the survival assay, and to Martin Harterink and Antoinette Killian for valuable discussions. References ↵ Alquezar , C. , Arya , S. , & Kao , A. W. ( 2021 ). Tau Post-translational Modifications: Dynamic Transformers of Tau Function, Degradation, and Aggregation . Frontiers in Neurology , 11 , 1 – 24 . doi: 10.3389/fneur.2020.595532 OpenUrl CrossRef ↵ Aquino Nunez , W. , Combs , B. , Gamblin , T. C. , & Ackley , B. D. ( 2022 ). Age-dependent accumulation of tau aggregation in Caenorhabditis elegans . 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Share Human tau compromises neuronal structural integrity in C. elegans promoted by age, stress and phosphorylation Maithili Joshi , Sanne van Falier , Tessa Sinnige bioRxiv 2025.08.01.668095; doi: https://doi.org/10.1101/2025.08.01.668095 Share This Article: Copy Citation Tools Human tau compromises neuronal structural integrity in C. elegans promoted by age, stress and phosphorylation Maithili Joshi , Sanne van Falier , Tessa Sinnige bioRxiv 2025.08.01.668095; doi: https://doi.org/10.1101/2025.08.01.668095 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7621) Biochemistry (17645) Bioengineering (13867) Bioinformatics (41873) Biophysics (21420) Cancer Biology (18550) Cell Biology (25443) Clinical Trials (138) Developmental Biology (13360) Ecology (19866) Epidemiology (2067) Evolutionary Biology (24289) Genetics (15587) Genomics (22472) Immunology (17707) Microbiology (40318) Molecular Biology (17142) Neuroscience (88457) Paleontology (666) Pathology (2826) Pharmacology and Toxicology (4815) Physiology (7634) Plant Biology (15111) Scientific Communication and Education (2042) Synthetic Biology (4285) Systems Biology (9813) Zoology (2268)