Nucleophagy delays aging and preserves germline immortality
Autophagy modulates nuclear architecture via ANC-1/nesprin-2
We investigated whether the C. elegans nesprin ortholog, ANC-1, regulates nuclear morphology during aging. Pronounced nuclear enlargement, shape abnormalities and lamin-1 (LMN-1) accumulation occur in the absence of ANC-1/nesprin-2, compared to WT animals and mouse cells (Fig. 1a,b and Extended Data Fig. 1a–d). Lamin B1 is a substrate of autophagy on oncogenic insult and exhibit increased protein levels in the absence of nesprin-215,16. Depletion of nesprin-2 causes lamin B accumulation in mouse embryonic fibroblasts (MEFs), which is mediated by blocking autophagy. Indeed, treatment with the autophagic flux inhibitor bafilomycin A1 (BafA1) increases lamin B abundance only in WT cells, while abnormal lamin B staining outside the nucleus is prominent in the absence of nesprin-2 (Extended Data Fig. 1e–g). Therefore, autophagic recycling of lamin B is, at least partially, regulated by nesprin-2. Thus, the role of nesprin family members in maintaining nuclear architecture is evolutionarily conserved from nematodes to mammals. Importantly, ANC-1 is essential and confers stress resistance against nutrient deprivation, heat and DNA damage (Extended Data Fig. 1h,i). Since autophagy is triggered as a cellular response mechanism to various stressors, we hypothesized that nesprin family members might be engaged in a selective type of nuclear autophagy to ultimately contribute to nuclear morphology homeostasis15,17 and stress resistance. To test this hypothesis, we first examined whether ANC-1 colocalizes with LGG-1 (microtubule associated protein 1 light chain 3 (LC3), gamma-aminobutyric acid receptor-associated protein (GABARAP) and GATE-16 family-1), the nematode autophagy-related protein 8/GABARAP protein homolog, on autophagy induction and autophagic flux inhibition by knockdown of insulin-like receptor subunit beta (daf-2) and Ras-related protein Rab-7 (rab-7), respectively (Fig.1c,d). Moreover, genetic inhibition of autophagy by lgg-1 and rab-7 RNA interference (RNAi) leads to accumulation of green fluorescent protein (GFP)::ANC-1 (Fig.1e,f). Similarly, autophagy-inducing conditions such as starvation and DNA damage (ultraviolet) modulate ANC-1 expression and nuclear area, as shown by using the mKate2::ANC-118 reporter (Fig. 1g,h). In MEFs, nesprin-2 localization is also altered on autophagic induction by DNA damage (Extended Data Fig. 2a), while nesprin-2-decorated nuclear protrusions form under DNA damaging conditions (etoposide and mitomycin C). Interestingly, these protrusions are analogous to the nuclear bulges formed during nucleophagic degradation of nuclear material15. In cortical neurons, nesprin-1 distribution is more punctate on autophagic flux inhibition (Extended Data Fig. 2b,c). On autophagic flux inhibition, the relative protein levels of the nesprin-2 ε1 isoform are elevated (Fig.1i), indicating that nesprin-2 is degraded via autophagy. Notably, we find that nesprin-2 expression increases under autophagy-inducing, starvation conditions as shown by monitoring relative RNA and protein levels (Extended Data Fig. 2d and Fig.1i). In addition, immunoprecipitation experiments indicate LC3 interaction with nesprin-1 and nesprin-2 (Extended Data Fig. 2e,f). Taken together, these findings suggest that nesprin-2 abundance is regulated by autophagy.
ANC-1/nesprin-2 regulates autophagosome formation
We next asked whether nesprin protein family members function as regulators of autophagy-related events, such as omegasome formation, autophagosome formation and autophagic protein degradation. Knockdown of the anc-1 gene, encoding the nematode nesprin ortholog, causes accumulation of double FYVE-containing protein 1 (DFCP1)::GFP puncta, signifying early autophagic structures19 in somatic tissues of adult C. elegans animals (Fig. 2a,b). Notably, depletion of ANC-1 under autophagy-related protein 2 (ATG-2) deficiency, a protein required for pre-autophagosomal structure organization, does not increase the number of DFCP1::GFP puncta further, indicating that both ANC-1 and ATG-2 function in the same pathway (Fig. 2a,b). We observed a similar elevation of LGG-1 puncta formation on ANC-1 depletion (Fig. 2a,b). We then assessed the requirement of ANC-1 under low insulin/insulin-like growth factor I (IGF-1) signaling, known to induce autophagy in daf-2 insulin/IGF-1 receptor mutant nematodes20,21,22. ANC-1 deficiency makes autophagosomal puncta larger, indicating that autophagosomes either increase in size or coalesce (Fig. 2a,b). Absence of ANC-1 induces accumulation of sequestosome related-1 (SQST-1), the C. elegans ortholog of p62 (Fig.2a,b). Genetic inhibition of early Beclin homolog 1 (bec-1) and late (rab-7) stage autophagy indicate that ANC-1 regulates late autophagic steps, since bec-1 downregulation showed further increase in GFP::LGG-1 accumulation compared to animals with anc-1 downregulation (Extended Data Fig. 3a,b). In addition, rab-7 downregulation failed to further accumulate GFP::LGG-1 compared to animals with anc-1 downregulation. Collectively, these observations indicate that ANC-1 is implicated in late autophagic steps; therefore, its absence affects progression and/or completion of the autophagic process. We used a polyglutamine (Q40) disease model, where polyglutamine aggregates form23. Autophagy has been shown to degrade such aggregate-prone proteins with polyglutamine expansion24,25. Remarkably, we show that young adult nematodes accumulate more puncta in the absence of ANC-1 (Fig. 2c,d). A similar phenotype was observed when ANC-1 and LGG-1 or BEC-1 were silenced in parallel, implying that ANC-1 suppresses the accumulation of polyglutamine aggregates through autophagy (Fig. 2c,d).
We then sought to corroborate the unexpected function of ANC-1 as an autophagy regulator in the mouse. To this end, we assessed the levels and distribution of LC3B and p62. Consistent with this notion, LC3B puncta were more abundant in nesprin-2−/− MEFs (Fig. 2e,f). Autophagic flux inhibition (BafA1) did not further increase LC3B puncta accumulation in nesprin-2−/− MEFs. In conjunction, in the absence of nesprin-2, the LC3II/I ratio exhibited an increased trend, albeit non-significant, indicating possibly defective autophagic clearance (Fig. 2g,h). In addition, LC3 puncta size was larger under autophagic flux inhibition, indicating autophagosome aggregation, when nesprin-2 was depleted (Fig. 2e and Extended Data Fig. 3c). p62 accumulated in the nucleus under basal conditions in the absence of nesprin-2 (Fig. 2i,j). Notably, we observed an aggregation of nuclear p62 (Fig. 2i,j), coupled with a nuclear and perinuclear accumulation of LC3B in the absence of nesprin-2 (Fig. 2e and Extended Data Fig. 3d–f), further suggesting a role of nesprin-2 in autophagy. Combined, these findings uncover an evolutionarily conserved role for nesprin protein family members in autophagy that could influence and, possibly, coordinate organelle-specific homeostasis, including the nucleus.
Autophagy and ANC-1/nesprin-2 control nucleolar homeostasis
Previous studies in diverse organisms, ranging from nematodes to humans, indicate that nucleolar size is a determinant of longevity under conditions of low insulin/IGF-1 signaling and dietary restriction9,26,27,28. However, the mechanisms responsible for nucleolar size regulation are elusive. Given the requirement of nesprins for autophagy under conditions of low insulin/IGF-1 signaling and nutrient deprivation, we investigated whether nesprin-mediated autophagy impinges on the nucleolus. Fibrillarin is a predominantly nucleolar protein, implicated in nucleolar size regulation7. Similarly to nesprin-2 and lamin A, it is found in nuclear bulges that have been implicated in nucleophagic degradation (Fig. 3a). Remarkably, we found that fibrillarin is a substrate of autophagy (Fig. 3b,c). Of note, depletion of nesprin-2 or blockage of basal autophagic flux in MEFs causes accumulation of fibrillarin and fibrillarin puncta expansion (Fig. 3b,c). We corroborated these findings by western blot analysis in nesprin-2-depleted MEFs where fibrillarin levels were increased on autophagic flux inhibition, albeit not significantly due to possible functional redundancy of nesprin-1 and 2 (Fig. 3d,e). Thus, nesprin-2 controls the amount of nucleolar fibrillarin via autophagy. In addition to fibrillarin, nesprin-2 modulates the expression of the 45S ribosomal RNA that is required for proper and efficient oocyte development, without interfering with the expression of core autophagy genes, such as LC3B (Fig. 3f). Lastly, we found that 5-month old, nesprin-2−/− mice displayed splenomegaly (Fig. 3g), which is a pathological condition related to lysosomal storage diseases29. These observations suggest that nesprins affect nucleolar size and function, likely through autophagic degradation of nuclear and nucleolar material.
To investigate the physiological significance of nesprin-2-mediated effects on the nucleolus in vivo, we monitored nucleolar structure perturbations in C. elegans, using a fibrillarin reporter strain (FIB-1::GFP). Notably, ANC-1 deficiency caused enlargement of nucleoli and increased endogenous FIB-1 protein levels, in otherwise WT animals, which is recapitulated by depletion of the C. elegans Beclin ortholog BEC-1 (Fig. 4a–c and Extended Data Fig. 4a,b). No further increase was observed by simultaneous knockdown of both ANC-1 and BEC-1, suggesting that they function in the same pathway to influence nucleolar size, while autophagic perturbation by either lgg-1 or rab-7 silencing alone or with simultaneous anc-1 silencing also increases FIB-1 protein levels (Fig. 4b,c and Extended Data Fig. 4a). By contrast, autophagy-inducing, low-insulin/IGF-1 signaling (on daf-2 knockdown) results in reduced nucleolar size and FIB-1 protein levels (Fig. 4a,c and Extended Data Fig. 4a). Importantly, either anc-1 or bec-1 knockdown is epistatic to DAF-2 deficiency (Fig. 4a,c,d). These observations indicate that ANC-1 and autophagy contribute downstream of insulin/IGF-1 signaling to preserve nucleolar morphology and homeostasis in C. elegans.
ANC-1 regulates somatic aging
Given the requirement of nesprin protein family members for autophagic recycling of nucleolar components such as fibrillarin, under both basal and nutrient stress conditions in diverse organisms, we examined whether nesprins also mediate the pro-longevity effects of low insulin/IGF-1 signaling and dietary restriction. Remarkably, knockdown of anc-1 shortened the life span of long-lived DAF-2-deficient nematodes, to an extent similar to that of BEC-1 depletion (Fig. 4d). Hence, ANC-1 functions to maintain nucleolar homeostasis and promote longevity under low insulin/IGF-1 signaling conditions. Similarly, loss of ANC-1 shortens the life span of long-lived caloric-restricted neuronal acetylcholine receptor subunit (EAT-2)-deficient animals (Extended Data Fig. 4c). FIB-1 silencing has been shown to increase life span; however, neither ANC-1 nor LGG-1 ablation further altered the life span, indicating that they act upstream of FIB-1 (Extended Data Fig. 4d). Moreover, fib-1(RNAi) does not increase the life span of long-lived daf-2 mutants (Extended Data Fig. 4d).
ANC-1 and autophagy promote gonad integrity and germline immortality
Strikingly, we observed distinct differences in proximal oocyte nucleolar size in the reproductive system of the nematode. Physiologically, small-sized proliferating germ cells in the gonad contain big nucleoli, which, as they enter meiosis and differentiate into oocytes, decrease their nucleolar size27. The most proximal oocyte, which will be fertilized, has no nucleolus. This process of regulating nucleolar size in the gonad is integral to germ cell differentiation and healthy oocyte production. To dissect this process, we examined ANC-1 expression in the gonad. ANC-1 colocalizes with fibrillarin in nucleoli (Extended Data Fig. 4e), while in the absence of ANC-1, proliferating gonadal germ cells have larger nucleoli (Extended Data Fig. 5a–c). Proximal oocytes fail to eliminate their nucleoli on ANC-1 or autophagic protein knockdown (Fig. 4e,f and Extended Data Fig. 5d,e). Autophagy controls nucleolar elimination at the proximal oocyte, as autophagy-deficient animals (lgg-2(tm5755), atg-18(gk378), lgg-1(RNAi) and bec-1(RNAi)), similar to the FIB-1 transcriptional repressor nucleolin (ncl-1) mutants, have prominent nucleoli at their proximal oocytes (Fig. 4e,f and Extended Data Fig. 5d,e) without impacting the rate of protein synthesis in vivo (Extended Data Fig. 5f)30. Worms grown at 25 °C lacking B-box type zinc finger protein NCL-1, autophagic components or ANC-1, display severe anatomical gonadal abnormalities after five generations and form tumor-like structures (Extended Data Fig. 5d and Fig. 4g). LGG-1 and ANC-1 colocalize in gonadal germ cells, indicating their functional interaction in the gonad (Fig. 4h,i). Notably, ANC-1-, autophagy- or NCL-1-deficient nematodes, which cannot shed their nucleoli in parallel to gonadal collapse, show progressive decline in fecundity under stress, with diminished egg laying capacity after five generations at 25 °C (Fig. 4j). Thus, nucleoli persistence at the most proximal oocytes seem to have physiological implications concerning germline immortality and act as a biomarker of gradual reduction in egg laying capacity. Apart from tumor formation, oocyte multinuclearity was also observed, similar to mammalian malignancies (Fig. 5a–c). Similar to tumors formed in the reproductive system of ANC-1-deficient hermaphrodite nematodes, female mice lacking nesprin-2, exhibit ovarian and endometrial tumors, showing evolutionary functional conservation (Fig. 4k)31. Analysis of ANC-1-depleted gonads across generations reveals gradual gonadal deterioration (Fig. 5d). Specifically, we observe degenerating features such as smaller aberrant oocytes (second generation), which later misalign with each other (third generation). Moreover, gonads exhibit malignancy-like phenotypes such as multinuclearity (third and fourth generation), tumor-like structures (fourth generation) and loss of cellular integrity (seventh generation) (Fig. 5d).
This quality control mechanism involving ANC-1 and LGG-1 is additive and synergistic with the core apoptotic cell death machinery. ANC-1 germ cell quality assurance can potentially act through a recently described autophagic cell death engulfment mechanism involving LGG-1, which has been shown to act cooperatively with the pro-apoptotic cell death abnormality protein 1 (CED-1) to degrade cells with accumulated damage (Fig. 5e and Fig. 6a–b)32. Corroborating ANC-1’s involvement in the clearance of dying cells, it colocalizes with LGG-1 under apoptosis-inducing conditions, either by silencing of prosurvival apoptosis regulator Bcl-2 ortholog CED-9 or by inducing DNA damage (ultraviolet) (Fig. 4h,i). Moreover, absence of LGG-1, ANC-1 or both causes accumulation of oocytes (Fig. 5e). We observed that on lgg-1 silencing, ANC-1 is localized to apoptotic corpses, either aggregating or forming a circular structure around the apoptotic corpses, which is reminiscent of the CED-1 pattern (Fig. 6c). Importantly, the gonads of worms grown at 25 °C for several generations show increased cellular damage in the absence of ANC-1, LGG-1, or both. They display gonads with abnormally large CED-1-decorated structures, which are indicative of aggregated germ cells/apoptotic corpses that cannot be cleared out due to their defective autophagic engulfment (Fig. 6d). These finding indicate that, similarly to DNA damage repair and chromosomal maintenance33,34, autophagic clearance of superfluous/damaged germ cells in the gonad may contribute to sustain germline immortality.