Putting Your Best Egg Forward

Abstract

In a recent issue of Nature, Bohnert and Kenyon (2017) describe a signaling pathway that prevents transgenerational inheritance of cytoplasmic protein aggregates. Fertilizing sperm trigger aggregate clearance in the ovum by a microautophagy-like effector mechanism mediated by inter-organelle communication between lysosomes and mitochondria.

In Jonathan Swift’s satire Gulliver’s Travels, the struldbrugs of Luggnagg live forever but continue to age, growing increasingly decrepit over the course of their endless lives. Fortunately for humans in the real world, natural selection (not generally known for its mercy) has spared us the horror of immortality without rejuvenation: our somatic tissues senesce and die, but the aging clock is reset with every new generation.

If the renewed youthfulness of children in comparison with their aged parents gives no solace to those who would stay forever young, it nonetheless speaks of the remarkable power of the germline to resist deleterious change over time. The integrity of the DNA is preserved by the high fidelity of replication and vigilant nucleic acid repair pathways. However, considerably less is known about the mechanisms germline cells use to maintain cytoplasmic homeostasis in the face of accumulated damage that threatens successful execution of the genetic blueprint. How, then, does the germline remain changeless, ensuring a perfect ovum every time?

In a recent study, Bohnert and Kenyon (2017) used oogenesis in the nematode Caenorhabditis elegans as a model to address this question. They focused on protein aggregation during the stepwise oocyte maturation process, whose temporal sequence is defined along the length axis of the gonad. They found that the cytoplasm of immature oocytes in the distal gonad are filled with protein aggregates, but that these are cleared following activation of lysosomes as maturing oocytes move proximally (and toward the spermatheca of these self-fertilizing hermaphrodites). This restoration of protein homeostasis (proteostasis) in the oocyte is dependent on a signal derived from sperm, ensuring that the resources required for perfection are only expended when fertilization is imminent.

Previous work showed that the level of oxidative protein damage is high in immature oocytes but drops as they mature (Goudeau and Aguilaniu, 2010). A laborious histological method was used to detect the damage, allowing only limited candidate testing of potential factors mediating this form of germline rejuvenation. The results obtained using this approach implicated the proteasome in the mechanism of damage clearance.

As a point of departure from this foundational work, Bohnert and Kenyon (2017) developed new live-cell fluorescent reporters of protein aggregation and used them to screen for proteostasis factors implicated in fertility. Using this less biased approach, they discovered that removal of aggregates requires components and activity of the V-ATPase—a proton pump that resides in the lysosome, an organelle capable of destroying cytosolic protein aggregates. As oocytes mature, V-ATPase levels rise, and the lysosomal interior becomes acidic, a sine qua non for the enzymatic activities of hydrolases within the compartment. A search for potential regulators of V-ATPase protein expression led the authors to analyze GLD-1, a pleiotropic translational repressor that is abundant in the distal, but not proximal, gonad. Genetic analysis revealed that GLD-1 represses translation of mRNAs encoding V-ATPase components and that the sperm signal induces degradation of GLD-1 by the proteasome (Figure 1).

Figure 1. Aggregate Clearance in C. elegans Oocytes.

(Top) Signaling pathway controlling the expression of V-ATPase subunits in C. elegans oocytes upon fertilization. Activation of a G protein (gray circle)-coupled receptor (gray serpentine shape) by sperm-derived ligands (yellow circles) leads to the acidification of lysosomes in mature oocytes. Acidified lysosomes engulf cytosolic protein aggregates (arbitrarily shown as ordered) by a microautophagy-like process. GLD-1 binds to the untranslated regions (UTRs) of mRNAs encoding V-ATPase subunits, repressing their translation (red bar-headed arrow). (Bottom) Metabolic and morphological changes that C. elegans oocyte mitochondria undergo as a result of lysosome acidification during fertilization. ROS, reactive oxygen species; J, electrochemical potential across the inner mitochondrial membrane. See Bohnert and Kenyon (2017) for additional experimental evidence supporting these working models. Figure by Charlene Chan.

The detailed mechanism underlying clearance of protein aggregates following lysosomal activation remains mysterious. However, the authors rule out one a priori likely explanation: macroautophagy, a form of cytoplasm-to-vacuole transport in which cargo fated for destruction is packaged into double-membrane vesicles that fuse with lysosomes. In many cell types, disruption of macroautophagy genes (ATGs) is associated with cytoplasmic protein aggregation (Mizushima and Levine, 2010), but Bohnert and Kenyon (2017) found that ATGs are dispensable for proper execution of their lysosomal switch. Curiously, real-time fluorescence microscopy revealed that activated lysosomes seem to extend protrusions toward aggregates fated for destruction, hinting that aggregates undergo direct invagination into the lysosome lumen by a process akin to microautophagy (Lefebvre et al., 2017). Forward genetic screens using the fluorescent protein aggregation reporters developed for this study could help answer pressing questions about the mechanisms underlying this cytoplasm-to-lysosome route: do protein aggregates enter the lysosome lumen in microautophagic vesicles? If so, is vesicle formation a selective process? And are ESCRT components involved in any associated membrane remodeling?

The authors continued by showing that lysosomal acidification leads to mitochondrial activation. As oocytes develop, their mitochondria also undergo morphological remodeling and functional changes. In immature distal oocytes, mitochondria are fragmented, generate high levels of reactive oxygen species (ROS), and have high membrane potentials, indicative of metabolic inactivity. Upon maturation, and dependent on sperm and functional V-ATPase, these mitochondrial phenotypes are reversed. This metabolic shift appears to be controlled by the oocyte’s energy charge (i.e., ATP/ADP ratio): microinjection of external ADP, which is both a V-ATPase product and a reactant of mitochondrial ATP synthase, was sufficient to bring about mitochondrial activation in immature oocytes.

What does this all have to do with aggregate clearance by lysosomes? Well, this is where things get a little baroque. Far from being a simple sequela of the lysosomal switch, the activated mitochondria actually give lysosomes a helping hand in aggregate clearance. The mechanistic details remain obscure, but increased aggregate mobility might be somehow involved.

An evolutionary and developmental perspective on the current work is provided by two previous studies of proteostasis during gametogenesis and transgenerational cytoplasmic inheritance. During replicative senescence in the budding yeast, the aging mother cell accumulates a variety of damage, including cytoplasmic protein aggregates (Unal et al., 2011). However, when an old diploid mother undergoes meiosis, it produces four haploid progeny whose reproductive ages have been reset. This rejuvenation process is associated with aggregate clearance by an undefined mechanism, which (like the one in the worm oocyte) requires lysosomal function. By contrast, in the mouse, oxidized protein damage is inherited during oogenesis and fertilization but is erased as embryonic cells differentiate and lose their pluripotency (Hernebring et al., 2006). To our knowledge, the potential role of lysosomes in this mechanism of aggregate clearance remains unexplored.

These findings leave plenty of fertile ground for speculation regarding both the physiological significance of aggregate clearance and the generality of the lysosome activity switch across species and cell types. The evolutionary benefit of proteostasis rejuvenation, in terms of the outcome for progeny, has yet to be demonstrated. What would happen if the germline did pass on its aggregated junk to the next generation (or if inherited junk persisted following cell differentiation)? It is reasonable to assume that the inherited detritus would decrease offspring fitness in some way, but this remains to be rigorously proven. If it were possible to engineer a worm capable of undergoing fertilization without triggering lysosomal activation, would its descendants exhibit progressively diminished vigor and ultimately become extinct?

The work of Bohnert and Kenyon (2017) reveals a fascinating mechanism for preventing transgenerational inheritance of cytoplasmic damage (Figure 1). It remains to be seen whether such a strategy is relevant outside the germline. Many types of somatic cells, especially neurons, accumulate protein aggregates over the course of aging, resulting in loss of function or viability. Could a lysosomal switch also serve to rejuvenate such aged cells? Intriguingly, in some long-lived worm mutants, the soma exhibit germline-like features (Curran et al., 2009). It will be interesting to investigate whether the germline lysosome switch is also activated in these mutants, conferring on them the extended vitality Swift’s cruel pen denied his immortal literary creations.