Cellular Senescence Is Induced by the Environmental Neurotoxin Paraquat and Contributes to Neuropathology Linked to Parkinson’s Disease
Summary
Exposure to the herbicide paraquat (PQ) is associated with an increased risk of idiopathic Parkinson’s disease (PD). Therapies based on PQ’s presumed mechanisms of action have not, however, yielded effective disease therapies. Cellular senescence is an anticancer mechanism that arrests proliferation of replication-competent cells and results in a pro-inflammatory senescence-associated secretory phenotype (SASP) capable of damaging neighboring tissues. Here, we demonstrate that senescent cell markers are preferentially present within astrocytes in PD brain tissues. Additionally, PQ was found to induce astrocytic senescence and an SASP in vitro and in vivo, and senescent cell depletion in the latter protects against PQ-induced neuropathology.
Our data suggest that exposure to certain environmental toxins promotes accumulation of senescent cells in the aging brain, which can contribute to dopaminergic neurodegeneration. Therapies that target senescent cells may constitute a strategy for treatment of sporadic PD, for which environmental exposure is a major risk factor.Introduction
Although genetic mutations that result in the development of familial Parkinson’s disease (PD) have been identified, these account for a minority of disease cases. The majority of cases are sporadic in nature and likely involve the contribution of various environmental exposures over the course of the lifespan (Wirdefeldt et al., 2011). A better understanding of the mechanisms involved in the contribution of these factors to neuropathology is a vital missing piece of the puzzle that will help unravel the underlying causes of idiopathic disease. Exposure to the herbicide paraquat (PQ) is strongly associated with increased lifetime risk for the development of sporadic PD. Its systemic administration in mice results in several hallmark neuropathological features of the disorder. However, therapies based on the agent’s presumed mechanisms of action have yet to yield effective disease therapies (Le et al., 2014, Wirdefeldt et al., 2011). Preventive and therapeutic treatments for idiopathic PD may require uncovering of disease mechanisms and targets distinct from those identified to date.
We report the contribution of a factor thought to contribute to aging in many tissues: cellular senescence (Campisi, 2013, Chinta et al., 2013, Chinta et al., 2015, Salminen et al., 2011). Cellular senescence is an anticancer mechanism occurring in cells capable of division, and it results in irreversible arrest of cell proliferation in response to stress that puts cells at risk for malignant transformation (Campisi, 2013, Prieur and Peeper, 2008). Senescent cells express a senescence-associated secretory phenotype (SASP)—the robust secretion of numerous inflammatory cytokines, chemokines, growth factors, and proteases (Coppé et al., 2008). Senescent cells accumulate with age in many human and mouse tissues. There, presumably by virtue of the SASP, they have been shown to contribute to the age-related loss of tissue structure and function (Baker et al., 2016, Campisi, 2013, Dimri et al., 1995, Muñoz-Espín and Serrano, 2014, van Deursen, 2014). Cellular senescence may be an example of evolutionary antagonistic pleiotropy—protecting organisms from cancer early in life but driving aging phenotypes at advanced age.
Although senescent cells have been demonstrated to contribute to age-related pathologies outside the brain, evidence to date for their contribution to age-related neurodegenerative diseases is only correlative (Bhat et al., 2012, Chinta et al., 2015, Mombach et al., 2015, Salminen et al., 2011, Turnquist et al., 2016). Here, we report that postmortem tissue from sporadic PD patients displays increased expression of senescent markers, particularly within astrocytes. PQ is also able to induce senescence in cultured human astrocytes and in vivo. Significantly, inducible depletion of in vivo senescence reduces the development of neurodegenerative phenotypes associated with systemic PQ exposure, demonstrating that senescent cells contribute to neurodegeneration in an in vivo neurodegenerative disease model. Our data suggest that strategies aimed at eliminating senescent cells hold promise as potential therapies to mitigate the development of sporadic PD, for which environmental stress is a major contributing factor.
Results
Senescent Astrocytes and SASP Factors Are Elevated in Parkinsonian Substantia Nigra Pars Compacta
Astrocytes expressing senescence markers increase in human brain tissue during normal aging (Kang et al., 2015) and further in affected brain regions of individuals with Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS) (Bhat et al., 2012, Turnquist et al., 2016), prompting us to ask whether senescent astrocytes are also present in PD (Figures 1A and 1B ). Compared to control tissues, Parkinsonian SNpc tissues displayed elevated expression of the senescence marker p16INK4a, and several SASP factors included the protease MMP-3 and the pro-inflammatory cytokines interleukin-6 (IL-6), IL-1α, and IL-8 (Figure 1A). Elevated p16INK4a and MMP-3 expression has also been reported in cortical tissue from AD patients (Bhat et al., 2012).
We next determined whether SNpc astrocytes in particular display evidence of cellular senescence in PD tissues. A reduced nuclear level of lamin B1, detectable by immunostaining in intact tissue, is an established senescence-associated marker (Freund et al., 2012). Astrocytes (GFAP+ cells) were found to be lamin B1 deficient in PD SNpc tissues while no significant difference was detected in PD versus aged-matched control tissues in non-astrocytic (GFAP−) neighboring cells (Figures 1B and S1). These findings suggest the elevated presence of senescence in affected PD tissues and that astrocytes may preferentially undergo senescence in diseased brain tissues.
PQ Induces Senescence and an SASP in Human iPSC-Derived Astrocytes
We next assessed the ability of human astrocytes derived from induced pluripotent stem cells (iPSCs) to undergo senescence in response to PQ, an environmental insult significantly associated with PD (Pezzoli and Cereda, 2013). In adult mice, chronic, low-dose systemic exposure to PQ recapitulates several important hallmarks of the disease, including selective loss of dopaminergic (DAergic) SNpc neurons and reduced motor function (McCormack and Di Monte, 2003, Pezzoli and Cereda, 2013, Tanner et al., 2011). Cultured human astrocytes exposed to PQ ceased proliferation based on decreased percentage of bromodeoxyuridine (BrdU)-labeled cells (Figure 2A). They also displayed several additional hallmarks of senescence, including increased senescence-associated β-galactosidase (SA-β-gal) activity (Dimri et al., 1995) (Figure 2B), increased levels of p16INK4a (Beauséjour et al., 2003) mRNA and protein (Figures 2C and 2D), and increased numbers of 53BP1 foci, the latter indicative of DNA-damage signaling, which is a potent stimulator of the SASP (Beauséjour et al., 2003)(Figure 2E). Consistent with development of an SASP, PQ also induced robust secretion of the pro-inflammatory cytokine IL-6 (Figure 2F), a prominent component of the SASP (Coppé et al., 2008).
Given that genotoxic stress induced by ionizing radiation (IR) initiates senescence with well-documented effects on mitosis and nuclear size (Pospelova et al., 2013), we verified that compared to IR, PQ caused a similar decrease in the percentage of Ki67+ cells (Figure 3A) and an increase in the mean size of astrocyte nuclei (Figure. 3B). Human fibroblasts cultured with PQ for 24 hr also displayed these phenotypes as well as decreased Lamin B1 levels and increased SA-β-gal activity (Figures 3A–3D). These data indicate that PQ is capable of inducing senescence in human astrocytes as well as human fibroblasts (Jung et al., 2009). However, astrocytes were more sensitive than fibroblasts to PQ-induced senescence based on SA-β-gal activity. Interestingly, a concentration of PQ that induced fibroblast senescence (>500 μM PQ) was lethal to astrocytes (Figure 3D).
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Soluble Factors Secreted by Senescent Human Astrocytes Compromise the Function of Neurons and Neural Progenitor Cells
Senescent cells can exert powerful paracrine effects on tissue microenvironments (Coppé et al., 2010); SASP factors have been shown to disrupt tissue structure and function (Brkic et al., 2015). We asked whether PQ-induced senescent astrocytes could drive PD-related neuropathologies associated with the neurotoxin, including DAergic neuronal cell death (Blesa and Przedborski, 2014) and reduced neural progenitor cell (NPC) proliferation and migration (Marxreiter et al., 2013). We cultured human DAergic neurons and NPCs in the presence of conditioned media (CM) from human astrocytes. CM from PQ-induced senescent astrocytes, but not non-senescent (vehicle-treated) astrocytes, significantly reduced the viability of DAergic neurons (Figure 5A). Similarly, CM from senescent astrocytes suppressed both NPC proliferation (Figure 5B) and migration (Figure 5C).
IL-6 is a prominent SASP factor, and elevated serum levels are associated with increased PD risk (Scalzo et al., 2010). Treating cultured DAergic neurons with recombinant IL-6 at the concentration found in the CM of PQ-treated astrocytes (1.5 μM) showed a trend toward reducing DAergic cell viability, but the effect was not statistically significant (Figure S2). This finding suggests that although IL-6 may contribute to neurotoxic SASP effects, additional factors are likely needed to mediate the detrimental impact on DAergic viability.
Depleting Senescent Cells Abrogates PQ-Induced Neuropathology
To test whether PQ-induced senescence contributes to neuropathologies in vivo, we used a mouse model that allows selective depletion of senescent cells (Demaria et al., 2014). These mice (p16-3MR) use the promoter for the senescence marker p16INK4a to drive expression of a modified herpes simplex virus thymidine kinase (HSV-TK), which converts the pro-drug ganciclovir (GCV) into a DNA chain terminator, causing cells to apoptose (Laberge et al., 2013). GCV eliminates 70%–80% of senescent cells in p16-3MR mice without discernible off-target or bystander effects (Demaria et al., 2014).
We subjected p16-3MR mice to a systemic PQ regimen shown to result in several neuropathological features of PD (McCormack and Di Monte, 2003, Shimizu et al., 2001). PQ increased nigrostriatal expression of p16INK4a and IL-6 (Figure 6A). As was the case for human astrocytes, PQ-treated p16-3MR astrocytes display increased SA-β-gal activity and were more sensitive to PQ-induced toxicity. Similar to astrocytes in human PD tissues, astrocytes from PQ-treated mice also displayed significant loss of nuclear lamin B1 and HMGB1 (high mobility group box 1), the latter necessary for SASP initiation (Figures 6B and 6C) (Davalos et al., 2013). Similar to our findings in the human PD SNpc, PQ did not significantly reduce nuclear lamin B1 in GFAP− cells (Figure 6D). Significantly, all senescent phenotypes in the SNpc were abrogated in mice in which senescent cells were eliminated by systemic GCV administration (Figures 6A–6D). We failed to detect a decline in the proliferation marker Ki67 in astrocytes in PQ-treated mice (Figure S3). However, Ki67 staining in astroglia in saline-treated animals was within the range of post-mitotic neurons and less than half the levels found in Ki67+ cells within the inner granule cell layer of the dentate gyrus, which are presumed to be actively dividing granule cell progenitors (Nicola et al., 2015).
Human PD neuropathology is characterized by the preferential loss of DAergic neurons in the SNpc, resulting in impairment of locomotor function (Jankovic, 2008). These phenotypes are recapitulated in the systemic PQ murine model. In the mouse SNpc, PQ caused a significant decline in a marker of DAergic neurons, tyrosine hydroxylase (TH); co-administration of GCV with PQ prevented this decline (Figure 7A). Systemic PQ administration is known to repress adult neurogenesis, but co-GCV administration abrogated this effect (Figure 7B) (Marxreiter et al., 2013). Finally, rearing behavior, a measure of motor neuron function more sensitive to DAergic cell loss than other motor tests (Willard et al., 2015), was significantly curbed in mice treated with PQ alone but normal in mice co-treated with GCV (Figure 7C). Taken together, these results suggest that senescent cell depletion abrogates PQ-mediated neuropathology and motor deficits.
Discussion
Late-onset idiopathic PD is thought to result from the combined effects of genetic risk factors, aging, and environmental exposure. Data from several laboratories suggest that humans and animals exposed to certain chemicals can develop neuropathological phenotypes associated with PD (Chinta et al., 2008, McCormack et al., 2005, Peng et al., 2010). One of the best studied of these is PQ. A meta-analysis of >100 epidemiological studies concluded that of several specific pesticides and chemical classes evaluated, only PQ was significantly associated with PD. Moreover, as this study points out, the epidemiologic data are backed by numerous animal studies demonstrating that PQ selectively depletes SNpc DAergic neurons and reduces motor function in adult mice, which are important hallmarks of human PD (Pezzoli and Cereda, 2013).
PQ is a free radical generator that causes oxidative stress (Bus and Gibson, 1984), which has been demonstrated to induce cellular senescence in replication-competent cell types (Campisi, 2013). PQ also results in compromised mitochondrial function, which has recently been tied to senescence induction in human fibroblasts (Wiley et al., 2016). One mechanism by which PQ may contribute to PD is by inducing cellular senescence within the brain (Chinta et al., 2013). Indeed, PQ induced senescence more readily in cultured human and murine astrocytes than in human fibroblasts, and their sensitivity was found to track with baseline SA-β-gal activity. Our studies suggest that PQ, like other inducers of senescence (Park et al., 2013), has a bandwidth of concentrations that induce senescence; above an upper limit, cells die. This finding raises the possibility that astrocytes are particularly prone to undergo senescence in response to chronic low-dose exposure to PQ that is associated with late-onset idiopathic PD. Indeed, administration of PQ to mice appears to preferentially increase senescent markers in astrocytes. Importantly, senescence markers were also elevated in astrocytes in SNpc tissues from autopsied PD brain samples, suggesting that the proclivity for astrocytes to undergo senescence may have physiological disease relevance. To our knowledge, this is the first report of elevated senescent markers in the PD brain.
While our data suggest that senescence occurs preferentially within astrocytes, this does not rule out senescence induction of other brain cell types. While neurons are post-mitotic and therefore unlikely to undergo traditional senescence induction, senescence has reported in other non-astrocytic cell types. For example, in response to repeated lipopolysaccharide administration, microglial BV2 cells display growth arrest, enhanced SA-β-gal activity, and heterochromatic foci (Yum et al., 2012). Based on our own findings that PQ can mediate senescence in disparate cell types (astrocytes and fibroblasts), we anticipate that at some concentrations PQ may induce senescence of other replicative brain cell types.
It is theoretically possible that senescence induction in peripheral tissues could contribute to PQ-mediated brain pathologies, but this seems unlikely, as the chronic, low-dose PQ regimen utilized in our studies is known to result in receptor-mediated uptake and immune response in the brain (McCormack and Di Monte, 2003) without activating peripheral immune cells or disrupting blood-brain barrier integrity (Vilas-Boas et al., 2014, Watson et al., 2013).
How might senescent astrocytes contribute to PD neuropathology? Mature astrocytes rarely divide in the adult brain (Ge et al., 2012, Sohn et al., 2015), consistent with our inability to detect Ki67+ astrocytes in the adult mouse brain; however, astrocytes do divide in response to certain injury (Barreto et al., 2011) and disease states (Boekhoorn et al., 2006, Bondolfi et al., 2002). The inability of senescent astrocytes to proliferate may exacerbate neuropathology when proliferation is an appropriate response to injury or disease. Astrocytes provide structural, metabolic, and trophic support to neurons (Allaman et al., 2011). When astrocytes adopt pro-inflammatory phenotypes, they can compromise neuronal function and contribute to age-related neurodegeneration and decrements in brain function in vivo (Rossi, 2015, Salminen et al., 2011). Factors secreted by senescent astrocytes had detrimental effects on both cultured human DAergic neurons and NPCs. CM from senescent astrocytes was supplemented with growth factors; therefore, trophic factor depletion seems an unlikely explanation. Secretion of IL-6 by cultured senescent human astrocytes was recently reported to decrease the viability of co-cultured motor neurons (Turnquist et al., 2016). We observed a similar increase in IL-6 secretion following PQ treatment in culture or in vivo, but IL-6 alone was not sufficient to induce significant degeneration of cultured DAergic neurons, suggesting that additional SASP factors may be required.
Why do DAergic SNpc neurons selectively degenerate in response to PQ and in PD? This is a pressing question in the field that our study does not explicitly address. However, we hypothesize that DAergic SNpc neurons may be particularly sensitive to the effects of oxidative stress on neighboring astrocytes, including senescence induction. In agreement with this idea, using an inducible astrocyte-specific oxidative stress mouse model, we previously demonstrated selective degeneration of DAergic SNpc neurons, despite astrocytes in other brain regions experiencing similar levels of oxidative stress (Mallajosyula et al., 2008).
In conclusion, our data show that astrocytes undergo cellular senescence in response to a well-established environmental stressor linked to PD and, significantly, that PQ-induced senescence contributes to PD-related neuropathology. This finding provide novel links among environmental exposure, cellular senescence, and PD. Further studies are warranted to identify other environmental and endogenous stressors capable of senescence induction in the brain. Our results suggest that senescent cells might be a novel therapeutic target for sporadic PD and other chronic neurodegenerative diseases in which environmental factors are implicated.
Acknowledgments
We thank Dr. John Trojanowski for providing materials from the University of Pennsylvania Brain Bank and Chris Lieu for assistance with behavioral studies. This work was funded by an Ellison Senior Scholar in Aging Award AG-SS-2620-11 (J.K.A.), Buck Impact circle (J.K.A and J.C), Michael J Fox Foundation, Target validation grant MJFF-12113 (J.K.A), NIH grant AG009909 (J.C.), an NIH Institutional Postdoctoral Training Grant T32-AG000266 (G.W.), a CIRM training grant TG2-01155 (S.J.C.), and the American-Italian Cancer Foundation (M.D.).
Author Contributions
S.J.C., G.W., J.K.A., and J.C. conceived the experiments and wrote the paper. S.J.C. and G.W., with the help of M.D., A.R., Y.Z., A.M., S.R., C.L., and D.T.M., conducted experiments and gathered data. S.J.C. and G.W. analyzed the data and prepared the figures.
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