Elevated lysosomal mass and enzyme activity in fibroblasts of the Mediterranean mouse Mus spretus

Elevated lysosomal mass and enzyme activity in fibroblasts of the Mediterranean mouse Mus spretus | 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 Elevated lysosomal mass and enzyme activity in fibroblasts of the Mediterranean mouse Mus spretus View ORCID Profile Melissa Sui , Joanne Teh , Kayleigh Fort , View ORCID Profile Daniel E. Shaw , View ORCID Profile Peter Sudmant , View ORCID Profile Tsuyoshi Koide , Jeffrey M. Good , View ORCID Profile Juan M. Vazquez , Rachel B. Brem doi: https://doi.org/10.1101/2025.02.05.636718 Melissa Sui 1 Departments of Plant and Microbial Biology Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Melissa Sui Joanne Teh 1 Departments of Plant and Microbial Biology Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kayleigh Fort 1 Departments of Plant and Microbial Biology Find this author on Google Scholar Find this author on PubMed Search for this author on this site Daniel E. Shaw 2 Division of Biological Sciences, University of Montana , Missoula, MT 59812, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Daniel E. Shaw Peter Sudmant 3 Integrative Biology, University of California , Berkeley, Berkeley, CA 94720, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Peter Sudmant Tsuyoshi Koide 4 National Institute of Genetics , Mishima, Shizuoka 411-8540, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tsuyoshi Koide Jeffrey M. Good 2 Division of Biological Sciences, University of Montana , Missoula, MT 59812, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Juan M. Vazquez 3 Integrative Biology, University of California , Berkeley, Berkeley, CA 94720, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Juan M. Vazquez For correspondence: juan{at}vazquez.bio rbrem{at}berkeley.edu Rachel B. Brem 1 Departments of Plant and Microbial Biology Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: juan{at}vazquez.bio rbrem{at}berkeley.edu Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract Failures of the lysosome-autophagy system are a hallmark of aging and many disease states. As a consequence, interventions that enhance lysosome function are of keen interest in the context of drug development. Throughout the biomedical literature, evolutionary biologists have found cases in which challenges faced by humans in clinical settings have been resolved by non-model organisms adapting to wild environments. Here, we used a primary cell culture approach to survey lysosomal characteristics in species of the genus Mus . We found that fibroblasts from M. spretus , a wild Mediterranean mouse, exhibited elevated lysosomal mass and enzyme activity along with reduced activity of β-galactosidase, a classical marker of cellular senescence, compared to those from M. musculus , a related species adapted to human-associated environments. We propose that classic laboratory models of lysosome function and senescence may reflect characters that diverge from the phenotypes of wild mice. The M. spretus phenotype may ultimately serve as a blueprint for interventions that ameliorate lysosomal dysfunction under conditions of stress and disease. Introduction Many aspects of metazoan health hinge on the ability of the lysosome-autophagy pathway to recycle damaged macromolecules and to direct cell growth decisions ( Shin & Zoncu 2020 ). Indeed, interventions that act broadly to promote health and longevity often require the lysosome-autophagy system ( Aman et al . 2021 ; Hansen et al . 2018 ; Bareja et al . 2019 ). More specific mechanisms to boost autophagy are also of potential clinical interest, particularly for treatment of proteinopathies and aging etiologies ( Bonam et al . 2019 ; Hansen et al . 2018 ). In practice, whether and how to stimulate proteostasis machinery to advance organismal health remains an open question, and the literature describing such manipulations is in its infancy ( Pyo et al . 2013 ; Leiva-Rodríguez et al . 2018 ; Shin et al . 2013 ; Liu et al . 2023 ; Bhuiyan et al . 2013 ; Sami et al. 2002 ; Bonam et al. 2019 ; Grabowski & Mistry 2022 ). Against a backdrop of decades of work in laboratory systems, ecologists have cataloged stress-and disease-resistance traits in wild genotypes from unusual niches, perhaps most famously in long-lived animal species ( Oka et al . 2023 ; Chusyd et al . 2021 ; Finch 2009 ). Cell-based surveys represent a powerful approach to find and dissect these natural resilience programs. One particularly fruitful discipline has profiled the variation in chemical stress resistance across primary cells from panels of non-model animals, including, in landmark cases, discovery of the underlying mechanisms ( Tian et al . 2019 ; Harper et al . 2007 ; Attaallah et al . 2020 ; Sulak et al . 2016 ). Cellular senescence has also been shown to vary across animal species in in vitro models ( Attaallah et al . 2020 ; Kang et al . 2023 ; Zhao et al . 2018 ; Gomes et al . 2011 ). In this study, we aimed to leverage species-specific variation in lysosomal markers within the Mus genus, to investigate how evolutionary processes have shaped the function of this organelle. Mouse species in this genus shared a common ancestor 7-8 million years ago, and as they radiated across Eurasia, M. musculus subspecies came to occupy human-associated niches, whereas other taxa are still found exclusively in the wild ( Pagès et al . 2015 ; Smissen & Rowe 2018 ). Our previous case study ( Kang et al . 2023 ) found differences in senescence behaviors, including lysosome markers, across fibroblasts from M. musculus subspecies and M. spretus , a wild relative that diverged 1-3 million years ago ( Morgan et al . 2022 ; Dejager et al . 2009 ). Here, we aimed to build on these observations to gain a deeper understanding of lysosomal programs in non-model mice, focusing on the contrasts between the genotypes of human commensal and laboratory mice and those of their wild relatives. Results M. spretus fibroblasts exhibit reduced lysosomal β -galactosidase activity We previously reported divergence among fibroblasts from several Mus genotypes ( Kang et al . 2023 ) in characteristics of cellular senescence, a stress-induced program of cell cycle arrest, resistance to apoptosis, and activation of immune signaling ( Campisi & d’Adda di Fagagna 2007 ; Campisi 2005 ; Hornsby 2002 ). Most salient from our initial findings were differences between Mus fibroblasts in activity of lysosomal β-galactosidase, which is used as a marker of senescence by virtue of its accumulation to detectable levels at suboptimal pH, due to the expanded lysosomal content of senescent cells ( Robbins et al . 1970 ; Magalhães & Passos 2018 ; Dodig et al . 2019 ; Curnock et al . 2023 ).To explore this variation further, we established a panel of primary tail skin fibroblasts from four wild-derived strains of M. m. musculus (PWK/PhJ, BLG2/Ms, CHD/Ms, MSM/Ms); two wild-derived strains of M. m. domesticus (ManB/NachJ, TUCA/NachJ); an admixed laboratory strain of mostly M. m. domesticus origin (C57BL/6J); a wild-derived strain of the steppe mouse M. spicilegus (ZRU); and two wild-derived strains of the Mediterranean mouse M. spretus (STF/Pas, SFM/Pas). We subjected each culture to 15 Gy of ionizing radiation and incubated for 10 days, which is sufficient to induce DNA damage response, arrested cell growth, and senescence expression programs in fibroblasts from across the genus ( Kang et al . 2023 , Hernandez-Segura et al . 2018 ). In assays of β-galactosidase, fibroblasts of each genotype displayed the anticipated increase in activity following irradiation, compared to their respective control counterparts ( Figure 1A-B and Figure S1 ). Whereas cells from strains of each species behaved similarly, we observed significant differences between species: irradiated M. spretus fibroblasts exhibited β-galactosidase signal ∼2-fold lower than did cells from M. m. musculus and M. m. domesticus, and staining of M. spicilegus cells was between the two extremes ( Figure 1A-B and Figure S1 ). To follow up on this difference, we used an alternative approach for senescence induction via the radiomimetic drug neocarzinostatin, which induces senescence in mouse cells ( Ito et al . 2018 ; Correia Melo et al . 2016 , Hernandez-Segura et al . 2018 ). Fibroblasts treated with neocarzinostatin exhibited a pattern mimicking that we had seen under radiation, with the treatment inducing elevated β-galactosidase activity in all cultures, and cells from M. spretus staining lower than those of sister taxa ( Figure 1C and Figure S1 ). Furthermore, the same trend was detectable in cells cultured in the absence of DNA damage: fibroblasts from M. musculus subspecies exhibited higher β-galactosidase staining than those of M. spicilegus , which stained higher than M. spretus . The latter species differences from untreated cells were of smaller magnitude than those manifesting after stress, and this dependence on treatment was statistically significant ( Figures 1 , S1 , and S2 ). Thus, M. spretus fibroblasts exhibited dampened β-galactosidase activity relative to other genotypes, with amplification of the effect under stress. Controls ruled out culture passage number as a driver of the variation ( Figure S3 ). Download figure Open in new tab Figure 1. Low β-galactosidase activity in M. spretus fibroblasts relative to M. musculus . In a given panel, in the plot at the top, each bar length displays the mean percentage of primary fibroblasts of the indicated strain and species that stained positive after administration of the colorimetric β-galactosidase substrate X-Gal. From top to bottom, complete names of the strains are as follows: M. m. musculus (PWK/PhJ, BLG2/Ms, CHD/Ms, MSM/Ms), laboratory strain of mostly M. m. domesticus origin (C57BL/6J), M. m. domesticus (TUCA/NachJ, ManB/NachJ), M. spicilegus , and M. spretus (SFM, STF/Pas). Representative images of primary fibroblasts from M. musculus PWK and M. spretus STF are shown at the bottom, with black arrows indicating dead cells. Panels report results from (A) untreated cells, (B) cells treated with ionizing irradiation followed by a 10-day incubation, or (C) cells after 1 hour of neocarzinostatin treatment followed by 24 hours of incubation. Points report biological and technical replicates collected over at least two separate days. Error bars report one standard error above and below the mean. ***, two-tailed Wilcoxon p < 10 -7 comparing M. spretus with all other genotypes; M. spretus and M. spicilegus also differed in each panel (Wilcoxon p < 0.05). For (B) and (C), in a comparison between the respective treatment and the untreated control in (A), a two-factor ANOVA with treatment and genotype as factors yielded p < 2e -16 for the interaction between the two factors. Replicate numbers are provided in Figure S12 . In principle, differences in the senescence-apoptosis fate choice after DNA damage ( Childs et al . 2014 ; Zhao et al . 2018 ; Attaallah et al . 2020 ) could contribute to the divergence between mouse genotypes that we had seen in fibroblast β-galactosidase staining. To explore this, we measured caspase 3/5 activity in fibroblasts in response to irradiation and neocarzinostatin treatment ( Figure S4 ). The results showed no consistent patterns of apoptosis either within or between species’ genotypes, arguing against a role for apoptosis activation in species differences in fibroblast β-galactosidase activity. To establish further the robustness of Mus species differences in fibroblast β-galactosidase activity, we considered the dose-response relationship with stress. We compared primary fibroblasts from M. m. musculus PWK and M. spretus STF as representatives of their respective species, in each case assaying β-galactosidase upon irradiation at increasing doses. The results revealed a consistent ∼2-fold difference between the genotypes at each dose ( Figure S5 ), a magnitude slightly exceeding the species divergence in the untreated control, consistent with our survey across genotypes at fixed stress doses ( Figure 1 ). These data ruled out a switch by M. spretus fibroblasts into a high-amplitude, M. musculus -like program above a certain threshold of stress exposure. We conclude instead that M. spretus cells are hard-wired for lower β-galactosidase activity in all tested conditions—including the resting state. To advance our search to understand Mus species differences in β-galactosidase activity in the fibroblast model, we next focused on regulation of the enzyme itself. Transcriptional profiling data from fibroblasts revealed 1.5 to 2-fold higher expression of the lysosomal β-galactosidase GLB1 in M. musculus fibroblasts than in those of M. spretus , regardless of treatment ( Figure S6A ). Allele-specific expression measurements in stressed and control fibroblasts from an interspecies F1 hybrid made clear that this change was regulated in cis : that is, the M. musculus allele of GLB1 drove higher expression of its own encoding locus than the M. spretus allele, when both were in the same nucleus ( Figure S6B ). Thus, the species changes in β-galactosidase activity we had noted in terms of cell biology in fibroblasts ( Figure 1 ) were mirrored by regulation of gene expression, including robust differences between genotypes in unstressed control conditions. No consistent divergence in senescence signaling across Mus species fibroblasts We reasoned that the differences in β-galactosidase activity in fibroblasts among the Mus species we tested were likely mediated by mechanisms unrelated to senescence itself. As a first investigation of this idea, we explored senescence signaling, using as a readout p21, a key regulator of cell cycle arrest in senescence ( Gu et al . 2013 ; Yew et al . 2011 ; Kreis et al . 2016 ). We quantified levels of p21 protein in response to either irradiation or neocarzinostatin, with fibroblasts from M. m. musculus PWK and M. spretus STF as a testbed. Results showed the expected induction of p21 in fibroblasts from both species in response to irradiation or neocarzinostatin ( Figure S7 ). In investigating quantitative patterns among these p21 induction behaviors, we noted some species-specific differences, though they were not of consistent direction: M. spretus cells induced p21 protein abundance more than did M. musculus under neocarzinostatin treatment but less following irradiation and in untreated controls ( Figure S7 ). These changes could reflect the differential activity of alternate senescence signaling pathways ( Saul et al . 2023 ) between species in some treatments. However, given our focus on lysosomal β-galactosidase differences between M. musculus and M. spretus across all tested conditions ( Figure 1 ), we concluded that regulation of p21 abundance was not a consistent correlate of this phenotype (although our results do not rule out p21 localization differences between the genotypes). Indeed, in analysis of mRNA expression profiles ( Kang et al . 2023 ), we detected no significant difference in induction of the p21 gene ( Cdkn1A ) between M. musculus and M. spretus fibroblasts in response to stress ( Figure S8 ). Increased lysosomal enzyme activity and lysosomal mass in M. spretus fibroblasts We hypothesized that the higher β-galactosidase expression and activity we had seen in M. musculus fibroblasts could be a consequence of heightened need owing to failures elsewhere in the proteostasis system, relative to cells of M. spretus . To explore this, we first assayed acidic compartments with the LysoTracker stain, again making use of M. m. musculus PWK and M. spretus STF as a test system. Conforming to our prediction, quantitative microscopy revealed a ∼2.5-fold increase in the cellular area stained by LysoTracker in untreated M. spretus fibroblasts compared with M. musculus ( Figure 2A ), and the species difference persisted in cultures induced to senesce with irradiation and neocarzinostatin ( Figure 2A ). A separate assay design using flow cytometry to quantify LysoTracker staining yielded similar results, with ∼3-fold higher levels in M. spretus cells in all conditions ( Figure S9 ). LysoTracker staining was absent from both M. musculus and M. spretus fibroblasts following treatment with the lysosomal H+-ATPase poison bafilomycin A1, confirming the dependence of this readout on lysosomal acidification ( Figure S10 ). Download figure Open in new tab Figure 2. Elevated lysosomal mass and enzyme activity in M. spretus fibroblasts relative to M. musculus . (A) Shown are results of quantitative microscopy analyses of staining by the lysosomal acidity reporter LysoTracker in primary fibroblasts of the indicated genotype ( M. spretus, strain STF; M. m. musculus , strain PWK). The y -axis reports the number of fluorescent pixels per cell, normalized to the control M. musculus sample for each experiment. Pairs of bars report results from untreated cells (left), or cells treated with irradiation followed by a 10-day incubation (middle) or after 1 hour of neocarzinostatin (NCS) treatment followed by 24 hours of incubation (right). At the bottom are shown representative fluorescence microscopy images of LysoTracker staining in primary fibroblasts from M. musculus PWK and M. spretus STF in untreated control, neocarzinostatin (NCS) treatment, and irradiation treatment. (B) Shown are results from assays of cathepsin D activity via BODIPY-pepstatin A fluorescence staining in fibroblasts of the indicated genotype ( M. spretus, strain STF; M. m. musculus , strain PWK). In the plot at the top, the y -axis reports the number of fluorescent pixels per cell, normalized to the control M. musculus sample for each experiment. Pairs of bars report results from untreated cells (left), or cells treated with irradiation followed by a 10-day incubation (middle) or after 1 hour of neocarzinostatin (NCS) treatment followed by 24 hours of incubation (right). At the bottom are shown representative fluorescence microscopy images of BODIPY-pepstatin A staining in primary fibroblasts from M. musculus PWK and M. spretus STF in untreated control, neocarzinostatin (NCS) treatment, and irradiation treatment.. Data points correspond to biological and technical replicates collected over at least two different days, and the bar height reports their mean. Error bars report one standard error above and below the mean. *, Wilcoxon p < 0.05 , **, Wilcoxon p < 0.01, ***, Wilcoxon p < 0.001. Replicate numbers are provided in Figure S12 . To investigate further the potential for enhanced lysosomal enzyme function in M. spretus cells compared to M. musculus , we developed an assay targeting the lysosomal enzyme cathepsin D. This assay employed a BODIPY-labeled pepstatin A conjugate, a fluorescent probe with established use in studies of lysosomal enzyme activity ( Boland et al . 2008 ; Lee et al . 2010 ). Measurements from experiments of this design made clear that M. spretus fibroblasts exhibited ∼2-fold higher area staining for cathepsin D activity than did M. musculus cells under untreated conditions, and similar though slightly dampened differences between the genotypes after irradiation or neocarzinostatin treatment ( Figure 2B ). Taken together, our results define a trait syndrome in M. spretus fibroblasts characterized by elevated lysosomal mass with acidic pH and enzymatic activity, relative to other Mus species, under both basal and senescent conditions. Lysosomal gene expression divergence between Mus fibroblasts To unearth clues to mechanisms of the divergence between Mus species fibroblasts in lysosomal characters, we revisited the expression programs of these cells, this time with a broad survey of lysosome-associated genes. The results revealed marked expression differences between purebred M. spretus and M. musculus fibroblasts ( Figure S11 ), including high expression in M. spretus cells of lysosomal biogenesis factors (namely Dtnbp1 and members of the Bloc1s family); the lysosomal regulator Lamtor1 ; and subunits of the lysosomal H+-ATPase domain ATP6v1 . Measurements of allele-specific expression in interspecies F1 hybrid fibroblasts established major contributions of cis -regulatory variation at many such genes ( Figure S11 ), meaning that the underlying genetic basis of a given gene’s expression change between purebreds could often be ascribed to heritable variants at the locus itself. We conclude that lysosomal gene regulatory changes between the fibroblasts of Mus species, controlled in part by cis -regulatory variants, parallel the divergence in lysosomal mass and enzyme activity— as expected if M. spretus cells made more lysosomes, and populated them to a greater extent with more functional enzymes, as a direct consequence of their distinct regulatory program. Discussion For decades, biomedical researchers have relied on strains from the M. musculus clade as the standard for studying lysosomal and senescence biology. In this work, we have demonstrated that lysosomal phenotypes vary quantitatively across Mus in fibroblast culture models, and that in contrast to M. musculus , cells from the non-commensal mouse M. spretus exhibit a program of lysosomal function of the kind that, in the experimental literature, has been associated with healthy aging and disease resistance. When experimentally induced in laboratory cell models, a backup in the lysosomal-autophagy system triggers compensatory increases in lysosomal mass and number, for which high β-galactosidase activity is a robust marker ( Curnock et al . 2023 ; Delfarah et al . 2021 ; Lee et al . 2006 , Dimri et al . 1995 , Rovira et al . 2022 ). We propose a similar causal relationship between the phenotypes we have observed as they vary across Mus fibroblasts. Under this model, the elevated β-galactosidase activity we have seen in M. musculus cells would represent compensation for their limited lysosomal mass and enzyme activity in comparison to M. spretus genotypes, even in the absence of stress. The M. musculus cis -regulatory allele upregulating β-galactosidase that we have noted in fibroblasts could well represent a genetically encoded component of such a program, a constitutive boost in protein degradation capacity by a regulatory mechanism, in the face of lysosomal defects. Indeed, we consider the broader set of lysosomal genes at which we have detected regulatory variation between the species as compelling candidate components of the mechanism underlying increased lysosomal mass and function in M. spretus cells. Our findings do leave open whether the compromised levels of these phenotypes in M. musculus cells represent an ancestral state in Mus that was resolved in the M. spicilegus–M. spretus lineage, or a derived trait that emerged alongside commensalism in M. musculus . In either case, the evidence that we have seen for conservation across M. musculus strains and subspecies strongly suggests a history of constraint in this lineage. If the lysosomal behaviors we have studied here prove to have evolved under selection, they could conceivably relate to body size, response to immune challenges, or association with human ecology as they differ between M. musculus and M. spretus ( Dejager et al . 2009 ; Mahieu et al . 2006 ; Kawakami & Yamamura 2008 ; Staelens et al . 2002 ; Blanchet et al . 2011 ; Pérez del Villar et al . 2013 ; Harr et al . 2016 ). Ultimately, as its mechanism is revealed, the elevated mass and enzyme activity of lysosomes that we have seen in M. spretus fibroblasts are of interest in the search for mimetics that would boost lysosomal function in a human clinical setting. More broadly, our results provide a way forward for the use of wild mouse species as models for lysosomal function and senescence, including future comparisons to the lysosomes of human cells and their phenotypes in disease. Methods Primary tail fibroblasts were extracted as described by Khan and Gasser (2016) . Subsequent culture and experiments used complete medium (DMEM, 10% FBS, 1% pen-strep) in T25 flasks at 37°C, 3% O 2 , and 10% CO 2 . Cells were passaged based on confluence using trypsin. Experimental treatments included 15 GY of ionizing irradiation or 3.6 µM neocarzinostatin. Analysis kits used were the Abcam Ltd. Senescence Detection Kit (Cat. #ab65351), ApoTox-Glo™ Triplex Assay (Promega Cat. #G6321), LysoTracker™ Green DND-26 (Thermo Fisher Cat. #L7526), and Pepstatin A BODIPY™ FL Conjugate (Thermo Fisher Cat. #P12271). Primary and secondary antibodies used in western blots were the rabbit monoclonal Anti-p21 antibody (ab188224, Abcam, 1:1000), mouse monoclonal anti-β-tubulin (T9026, Sigma-Aldrich, 1:1000), Goat Anti-Mouse IgG(H+L) Human ads-HRP (Cat#1031-05, Southern Biotech, 1:5000), and Goat Anti-Rabbit IgG(H+L) Human ads-HRP (Cat#4050-05, Southern Biotech, 1:5000). Additional details of methods available in Supplementary Materials. Data Accessibility Statement Data used in this manuscript can be found in an external data repository on Figshare at https://doi.org/10.6084/m9.figshare.c.8086870.v1 . RNA-sequencing data used in this manuscript can be found in the NCBI Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/ ) under accession number GSE201217. Supplementary figure captions Download figure Open in new tab Figure S1. Impact of genotype and treatment on β-galactosidase activity in Mus fibroblasts. Data are those of Figure 1 of the main text, grouped by genotype to emphasize treatment effects. A given set of three bars represents results from primary fibroblasts of the indicated strain from three conditions: untreated cells (darkest color), cells exposed to ionizing irradiation followed by a 10-day incubation (medium color), and cells treated with neocarzinostatin for 1 hour followed by 24 hours of incubation (lightest color). Download figure Open in new tab Figure S2. Low normalized β-galactosidase activity in M. spretus fibroblasts. Data are as in Figure 1 of the main text, except that measurements from the culture of a given genotype and treatment were normalized to the average of all measurements from untreated controls of that genotype. ***, two-tailed Wilcoxon p < 0.001 comparing M. spretus with all other genotypes for both treatments. Download figure Open in new tab Figure S3. Fibroblast passage number has no detectable effect on X-Gal staining. In a given panel, each row reports results from assays of the β-galactosidase substrate X-Gal on fibroblasts of the indicated genotype shown in Figure 1 of the main text, and each point reports one replicate culture. The x-axis reports the passage number of the respective culture and the y -axis reports X-Gal staining. (A) Cells induced to senesce with irradiation (right) and their paired controls (left). (B) Cells induced to senesce with neocarzinostatin (right) and their paired controls (left). Replicate numbers are provided in Figure S12 . Download figure Open in new tab Figure S4. No consistent variation between Mus species fibroblasts in apoptosis activity. Data are as in Figure 1 of the main text, except that for a given genotype and treatment, the x -axis reports caspase activity of the culture measured with the Apotox-Glo assay, normalized to the average from untreated cultures of the respective genotype for each experiment. (A) Outliers trimmed for visualization. (B) Full data set view. Replicate numbers are provided in Figure S12 . Download figure Open in new tab Figure S5. Dose dependence of β-galactosidase staining in Mus fibroblasts. Each trace shows results from assays of the β-galactosidase substrate X-Gal on primary fibroblasts of the indicated genotype ( M. spretus , strain STF; M. m. musculus , strain PWK). For a given trace, the x-axis reports irradiation dosage (in centigray, cGy) at 0 (Control), 1000, 1500, 2000, and 2500 cGy, and the y-axis reports the percentage of X-Gal positive cells in untreated controls or in irradiated cells after 10 days of incubation. Smaller points represent biological and technical replicates, and larger points indicate their mean values. ***, two-factor ANOVA with dosage and species as factors yielded p < 2e -16 for each factor. Replicate numbers are provided in Figure S12 . Download figure Open in new tab Figure S6. Variation between Mus fibroblasts in Glb1 mRNA expression. (A) Shown are measurements of mRNA expression of the β-galactosidase gene from profiling of fibroblasts of the indicated genotype ( M. spretus , strain STF; M. m. musculus , strain PWK), untreated or treated with irradiation followed by a 10-day incubation ( Kang et al . 2023 ). Data points within each column represent biological and technical replicates and the bar height reports their mean. Only the effect of genotype was significant in a two-factor ANOVA with condition and species as factors (*, p < 0.05). Wilcoxon test yielded no significant difference between the species for each condition. (B) Data are as in (A), except that allele-specific mRNA expression of the β-galactosidase gene was measured from profiling of fibroblasts of an F1 hybrid between M. spretus , strain STF and M. m. musculus , strain PWK ( Kang et al . 2023 ). In a two-factor ANOVA with condition and genotype as factors, both had significant effects (species p < 0.0005, ***; condition p < 0.05, *). Replicate numbers are provided in Figure S12 . Download figure Open in new tab Figure S7. No consistent variation between Mus fibroblasts in p21 abundance. Shown are results of Western blot assays of abundance of the senescence regulator p21 in primary fibroblasts of the indicated genotype ( M. spretus , strain STF; M. m. musculus , strain PWK). (A) Top, representative blot showing tubulin (55 kDa) and p21 (21 kDa) levels in 25 µg of whole cell lysate (WCL) from untreated control cells or those irradiated (Xray) and incubated for 10 days to establish senescence. Bottom, blot as at top except that cells were treated with 1 hour of neocarzinostatin (NCS) and incubated for 24 hours to establish senescence. (B) Each column reports quantification of p21 protein abundance normalized to tubulin for the indicated genotype and treatment. Data points correspond to biological and technical replicates collected over at least two different days, and the bar height reports their mean. Error bars report one standard error above and below the mean. No comparisons were significant in pairwise Wilcoxon tests. *, two-factor ANOVA with condition and species as factors yielded p < 0.05 for species. Replicate numbers are provided in Figure S12 . Download figure Open in new tab Figure S8. No significant variation between Mus fibroblasts in Cdkn1a mRNA expression. Shown are measurements of mRNA expression of the p21 gene Cdkn1a, from profiling of fibroblasts of the indicated genotype ( M. spretus , strain STF; M. m. musculus , strain PWK), untreated or treated with irradiation followed by a 10-day incubation ( Kang et al . 2023 ). Data points within each column represent biological and technical replicates and the bar height reports their mean. Error bars report one standard error above and below the mean. Only condition had a significant impact on expression in a two-factor ANOVA with treatment and genotype as factors (p < 0.005, **). Replicate numbers are provided in Figure S12 . Download figure Open in new tab Figure S9. Fluorescent signal quantification of LysoTracker staining indicates enhanced intracellular acidity in M. spretus fibroblasts. Shown are results from assays of the intracellular acidity reporter LysoTracker on primary fibroblasts of the indicated genotype ( M. spretus, strain STF; M. m. musculus , strain PWK). The y -axis reports mean fluorescence of the indicated culture normalized to the control M. musculus sample for each experiment. Data points correspond to biological and technical replicates collected over at least two different days, and the bar height reports their mean. Error bars report one standard error above and below the mean. *, Wilcoxon p < 0.05 , **, Wilcoxon p < 0.01. Replicate numbers are provided in Figure S12 . Download figure Open in new tab Figure S10. Bafilomycin A1 treatment abrogates LysoTracker fluorescent staining. (A) Shown are representative microscopy images of intracellular acidity reporter LysoTracker in untreated primary fibroblasts of the indicated genotype ( M. spretus, strain STF; M. m. musculus , strain PWK) after bafilomycin A1 treatment. (B) Alamar Blue cell viability stain results are shown for primary fibroblasts of the indicated genotypes ( M. spretus , strain STF; M. m. musculus , strain PWK) either with or without bafilomycin A1 treatment, or a no-cell control (Blank). The y-axis indicates absorbance of cell cultures at 570 nm, corresponding to Alamar Blue after reduction by active cellular metabolism. Data points within each column represent technical replicates and the bar height reports their mean. Download figure Open in new tab Figure S11. Variation between species fibroblasts in lysosomal gene expression. Each point reports the effect of genetic variation between M. spretus STF and M. m. musculus PWK on the expression of a lysosomal gene in primary skin fibroblasts from ( Kang et al . 2023 ). The x -axis shows the ratio of expression of the M. spretus allele relative to that of the M. musculus allele in an F1 hybrid background, indicating the contribution of cis -acting regulatory variants. The y -axis displays gene expression in purebred M. spretus fibroblasts normalized to that of M. musculus . Genes positioned near the diagonal line exhibit expression differences primarily driven by cis -regulatory variation. Red-labeled genes are highlighted in the main text. Download figure Open in new tab Figure S12. Sample sizes. Shown are sample sizes for each experiment presented in the main and supplementary figures. (n) denotes the number of independent animals used per experiment, (c) denotes the number of independent culture replicates used per experiment, and (q) denotes the number of cells quantified in the experiment. Acknowledgements The authors thank Vera Gorbunova, Emilie Tu, Helen Bateup, Samantha Jackson, Linda Wilbrecht, Polly Campbell, and Michael Nachman for animal material; Diana Bautista, Britt Glaunsinger, José Pablo Vázquez-Medina, and Mary West for their generosity with space and resources; Juliana Valencia Lesmes, Mara Baylis, Sam Rider, Harriet Song and William D’Angelo for technical contributions; and members of the Brem lab for helpful discussions. This work was supported by National Institutes of Health R01NS116992 to RBB and R01GM120430 to RBB and JMG. JMV was supported by the National Science Foundation Postdoctoral Research Fellowship in Biology (#2109915) and by the National Institute for Health T32AG000266 and 1K99AG088361. 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Share Elevated lysosomal mass and enzyme activity in fibroblasts of the Mediterranean mouse Mus spretus Melissa Sui , Joanne Teh , Kayleigh Fort , Daniel E. Shaw , Peter Sudmant , Tsuyoshi Koide , Jeffrey M. Good , Juan M. Vazquez , Rachel B. Brem bioRxiv 2025.02.05.636718; doi: https://doi.org/10.1101/2025.02.05.636718 Share This Article: Copy Citation Tools Elevated lysosomal mass and enzyme activity in fibroblasts of the Mediterranean mouse Mus spretus Melissa Sui , Joanne Teh , Kayleigh Fort , Daniel E. Shaw , Peter Sudmant , Tsuyoshi Koide , Jeffrey M. Good , Juan M. Vazquez , Rachel B. 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