Abstract
Exosomes are membrane-bound vesicles released by many cells including neurons, carrying cargoes involved in signaling and disease. It has been unclear whether exosomes promote intercellular signaling in vivo or serve primarily to dispose of unwanted cargo. This is because manipulations of exosome cargo expression or traffic often result in their depletion from the donor cell, making it difficult to distinguish whether these cargoes act cell-autonomously or through transcellular transfer. Exosomes arise when multivesicular endosomes fuse with the plasma membrane, releasing their intralumenal vesicles outside the cell. We show that loss of multivesicular endosome-generating ESCRT (endosomal sorting complex required for transport) machinery disrupts release of exosome cargoes from Drosophila motor neurons, without depleting them from the donor presynaptic terminal. Cargoes and autophagic vacuoles accumulate in presynaptic terminals, suggesting that compensatory autophagy follows endosome dysfunction. Surprisingly, exosome cargoes Synaptotagmin-4 (Syt4) and Evenness Interrupted (Evi) retain many of their signaling activities upon ESCRT depletion, despite being trapped in presynaptic terminals. Thus, these cargoes may not require intercellular transfer, and instead are likely to function cell autonomously in the motor neuron. Our results indicate that synaptic exosome release depends on ESCRT, and serves primarily as a proteostatic mechanism for at least some cargoes.
Introduction
Neurons release extracellular vesicles (EVs) that can mediate intercellular communication, dispose of unwanted neuronal components, and propagate pathological factors in neurodegenerative disease (Budnik et al., 2016; Holm et al., 2018; Song et al., 2020). Many elegant functional studies of neuronal EVs involve their purification from donor cells and subsequent application to target cells for tests of biological activity (e.g. Gong et al., 2016; Vilcaes et al., 2021). These experiments demonstrate that EVs containing specific cargoes are sufficient to cause functional changes in the recipient cell, but do not rigorously show that traffic into EVs is necessary for the functions of cargoes in vivo. In the donor cell, EV cargoes are typically trafficked through the secretory system, plasma membrane, and endosomal network, where they might execute intracellular activities before being released (van Niel et al., 2018). Therefore, to test the physiological functions of EVs in vivo, it will be essential to uncouple potential donor cell-autonomous from transcellular functions of these cargoes, using tools that specifically block EV release. Developing such tools will require a deeper understanding of how cargoes are packaged into EVs, and released in a spatially and temporally controlled fashion, especially within the complex morphology of neurons (Blanchette and Rodal, 2020).
Exosomes are a type of EV that arise when multivesicular endosomes (MVEs) fuse with the plasma membrane, releasing their intralumenal vesicles (ILVs) into the extracellular space. Spatial and temporal regulation of the machinery that controls formation of MVEs is therefore likely to be critical for exosome cargo selection, packaging, and release. MVEs can form via multiple nonexclusive mechanisms for budding of vesicles into the endosomal lumen (van Niel et al., 2018). One such pathway relies on Endosomal Sorting Complex Required for Traffic (ESCRT) proteins. In this pathway, ESCRT-0, -I, and -II components cluster cargoes, deform membranes, and then recruit ESCRT-III components, which form a helical polymer that drives fission of the ILV. Finally, the VPS4 ATPase recycles ESCRT-III filaments (Gruenberg, 2020; Vietri et al., 2020). The ESCRT-I component Tsg101 (Tumor susceptibility gene 101) is incorporated into and serves as a common marker for EVs, highlighting the link between ESCRT and EVs (van Niel et al., 2018). A neutral sphingomyelinase (nSMase)-mediated pathway may operate together with or in parallel to ESCRT to generate EVs by directly modifying lipids and altering their curvature, and indeed EV release of many neuronal cargoes is sensitive to nSMase depletion or inhibition (Asai et al., 2015; Dinkins et al., 2016; Goncalves et al., 2015; Guo et al., 2015; Men et al., 2019; Sackmann et al., 2019). The ESCRT machinery also has functions beyond MVE formation, including autophagosome closure and organelle repair, which are in turn involved in alternative modes of EV biogenesis (Arbo et al., 2020; Lefebvre et al., 2018; Leidal and Debnath, 2021; Moreau et al., 2013; Zhen and Stenmark, 2023). However, there is evidence both for and against a role for ESCRT in EV biogenesis in different neuronal cell types (Cone et al., 2020; Coulter et al., 2018; Gong et al., 2016). Thus, it remains unclear if or how EV dysfunction contributes to organism-level physiological defects resulting from ESCRT disruption, including ESCRT-linked human neurological disease (Dubey et al., 2022; Filimonenko et al., 2007; Ugbode and West, 2021; Willén et al., 2017; Yan and Zheng, 2021).
At the Drosophila larval neuromuscular junction (NMJ), EVs are released from presynaptic motor neurons into extrasynaptic space within the muscle membrane subsynaptic reticulum, and can also be taken up by muscles and glia (Fuentes-Medel et al., 2009; Koles et al., 2012). These EVs are likely to be exosomes, as cargoes are found in presynaptic MVEs, and depend on endosomal sorting machinery for their release and regulation (Blanchette et al., 2022; Koles et al., 2012; Korkut et al., 2009; Lauwers et al., 2018; Walsh et al., 2021). This system provides the powerful advantage of investigating endogenous or exogenous exosome cargoes with known physiological functions in their normal tissue and developmental context. Cargoes characterized to date include Synaptotagmin-4 (Syt4, which mediates functional and structural plasticity), Amyloid Precursor Protein (APP, a signaling protein involved in Alzheimer’s Disease), Evenness Interrupted/Wntless/Sprinter (Evi, which carries Wnt/Wingless (Wg) to regulate synaptic development and plasticity), and Neuroglian (Nrg, a cell adhesion molecule) (Korkut et al., 2013, 2009; Walsh et al., 2021). Mutants in trafficking machinery (for example evi and the recycling endosome GTPase rab11) cause reduced levels of cargo in EVs and show defects in EV cargo physiological activities, leading to the hypothesis that trans-synaptic transfer of these cargoes into the postsynaptic muscle is required for their signaling functions (Budnik et al., 2016). However, we and others have shown that these mutants also have a dramatic local presynaptic decrease in cargo levels, making it difficult to rule the donor neuron out as their site of action (Ashley et al., 2018; Blanchette et al., 2022; Koles et al., 2012; Korkut et al., 2009; Walsh et al., 2021). Here we show that disruption of the ESCRT machinery causes a specific loss of EV release without depleting presynaptic cargo levels, and use this system to test whether exosome release is required for cargo signaling functions.
Results
ESCRT machinery promotes EV release from synapses
To determine if the ESCRT pathway is involved in EV release at the Drosophila NMJ synapse, we first used GAL4/UAS-driven RNAi to knock down the ESCRT-I component Tsg101 (Tumor susceptibility gene 101) specifically in neurons (Tsg101KD). We then used our previously established methods to measure the levels of the endogenously tagged EV cargo Syt4-GFP, both in the donor presynaptic compartment and in neuron-derived EVs in the adjacent postsynaptic cleft and muscle (Walsh et al., 2021). Neuronal knockdown of Tsg101 (Tsg101KD) led to accumulation of Syt4-GFP in the presynaptic compartment, together with a striking loss of postsynaptic Syt4-GFP EVs (Fig 1A, E). We next tested the effects of Tsg101KD on three other known EV cargoes: UAS-driven Evi-GFP (1B, F) or human APP-GFP (1C, G), and endogenous Nrg (1D, H) (Korkut et al., 2013, 2009; Walsh et al., 2021). For all three cargoes, we observed a similar phenotype as seen for Syt4-GFP: presynaptic redistribution in large structures (accumulating to particularly high levels for Syt4-GFP and Evi-GFP), together with loss of postsynaptic EV signal. Thus, multiple EV cargoes, either endogenously or exogenously expressed, require the ESCRT-I component Tsg101 for release in neuronally-derived EVs. Further, since cargoes accumulate in internal structures upon Tsg101KD, these data suggest that NMJ EVs are derived from intracellular MVEs rather than budding directly from the plasma membrane, so we will refer to them as exosomes for the remainder of this study.
In addition to its functions in MVE biogenesis, Tsg101 also plays roles in numerous cellular processes including membrane repair, lipid transfer, neurite pruning, and autophagy, each depending on a specific subset of other ESCRT machinery (Vietri et al., 2020). We therefore tested if exosome release depends on other canonical ESCRT components. Hrs (Hepatocyte growth factor receptor substrate) is a component of the ESCRT-0 complex and is required to cluster exosome cargo on the delimiting membrane of the endosome (Vietri et al., 2020). Similar to Tsg101KD, Hrs loss-of-function mutants caused a drastic decrease in postsynaptic Nrg, though interestingly presynaptic Nrg was also partially depleted, unlike the Tsg101KD condition (Fig 1I, J). Next, we tested ESCRT-III, which forms the polymer involved in constriction and scission of the ILV neck. The Drosophila genome encodes several ESCRT-III proteins, of which shrub is homologous to mammalian CHMP4B. Shrub is likely to play an important role at synapses, since its loss leads to defects in NMJ morphogenesis and ILV formation (Sweeney et al., 2006). Pan-neuronal RNAi of shrub (ShrubKD) caused a dramatic loss of postsynaptic Syt4 and Nrg signals (Fig. 1K-M). Finally, we examined the role of Vps4, which catalyzes disassembly of the ESCRT-III polymer, finalizing the formation of the ILV. Pan-neuronal expression of a dominant negative Vps4 fragment (Vps4DN, (Rodahl et al., 2009)) strongly reduced postsynaptic levels of both Syt4-GFP and Nrg, and increased their presynaptic levels (Fig. 1N-P). These results demonstrate that multiple components of the ESCRT pathway are required for release of exosome cargoes at neuronal synapses, with variable effects on presynaptic accumulation of these cargoes.
Loss of Tsg101 leads to accumulation of cargoes in arrested autophagic structures
To explore the nature of the presynaptic accumulations of exosome cargoes at Tsg101KD NMJs, we examined their colocalization with early (Rab5) and recycling (Rab11) endosomes, which drive an endosome-to-plasma membrane recycling flux that supplies the exosome biogenesis pathway at this synapse (Korkut 2009, 2014, Walsh 2021). We also examined cargo co-localization with late endosomes (Rab7), which play less important roles in NMJ exosome traffic (Walsh 2021). We found that exosome cargoes exhibited increased co-localization with all these endosomal markers at Tsg101KD synapses, in what appeared to be multi-endosome clusters (Fig 2A, S1A-C). These results argue against formation of a single type of arrested MVE such as the canonical Class E compartment in ESCRT-deficient yeast and mammalian cells (Doyotte et al., 2005; Raymond et al., 1992) and instead suggest a more global defect in endosome maturation or turnover. To test this hypothesis, we measured the overall mean intensity as well as puncta number and size for Rab5, Rab7, and Rab11 upon Tsg101 knockdown. We saw no changes in the total intensity or puncta parameters for Rab7 (Fig S1D-F). However, for both Rab5 and Rab11, we observed a significant increase in Rab puncta intensity, mean intensity over the whole NMJ, and an increase in puncta size, with no change (Rab11) or a slight decrease (Rab5) in the number of puncta (Fig S1D-F). These results suggest defects in early and recycling endosome maturation and/or turnover upon loss of ESCRT function.
We next examined Tsg101KD NMJs using transmission electron microscopy (TEM). Tsg101KD boutons retained typical mitochondria, synaptic vesicles and active zone “Tbars”, and were surrounded by an apparently normal subsynaptic reticulum, representing the infolded postsynaptic muscle membrane. However, within Tsg101KD boutons we observed striking clusters of double membrane-surrounded or electron-dense structures, typical of autophagic vacuoles at various stages of maturation (Klionsky et al., 2021), including those with unclosed phagophores (Fig. 2B; three or more autophagic vacuoles were observed in 58.9% of mutant boutons (n=56) compared to 2.5% of control boutons (n=40), p<0.0001). Given that secretion of autophagosomal contents has been described as an EV-generating mechanism (Buratta et al., 2020), we next tested whether core autophagy machinery might play a role in exosome release at the Drosophila NMJ, and could therefore be linked to the Tsg101KD exosome phenotype. Atg1 is a kinase that is required for the initiation of phagophore assembly, and acts as a scaffold for recruitment of subsequent proteins, while Atg2 is required for phospholipid transfer to the phagophore (Nakatogawa, 2020). We observed a modest but significant decrease in both pre- and post-synaptic levels of the exosome marker Nrg upon disruption of autophagy by RNAi-mediated knockdown of Atg1 (knockdown validated in Fig. S2A, Nrg results in Fig. S2B-C), as well as by loss-of-function Atg2 mutations (Fig. S2D-E). Notably, these mutants did not recapitulate the ESCRT mutant phenotype of dramatic depletion of postsynaptic exosomes and presynaptically trapped cargoes. These results indicate that autophagic machinery does not play a major role in exosome biogenesis or release at the NMJ, and that the autophagic defects at Tsg101KD NMJs are likely separable from its roles in exosome release.
To further explore these autophagic defects, we next used the reporter GFP-mCherry-Atg8/LC3 to assess autophagic flux in Tsg101KD neurons. Under normal circumstances, the GFP moiety in this reporter is quenched when autophagosomes fuse with acidic endosomes and lysosomes, while mCherry retains its fluorescence. By contrast, defects in autophagic flux lead to accumulation of structures with both GFP and mCherry fluorescence (Klionsky et al., 2021). We examined this flux in motor neuron cell bodies, where mature autolysosomes are predicted to accumulate (Sidibe et al., 2022). In wild-type animals we observed mCherry-positive/GFP-negative puncta reflecting mature autolysosomes. By contrast we observed an increased volume of intense puncta in the cell bodies of Tsg101KD motor neurons, most of which were labeled by both mCherry and GFP (Fig. 2C, D, S2F). These data suggest that Tsg101KD causes a defect in autophagic flux.
Since autophagy is normally rare at wild-type Drosophila NMJ synapses (Soukup et al., 2016), we hypothesized that ESCRT mutants might activate a compensatory “endosomophagy” pathway to degrade ESCRT-deficient endosomes (Millarte et al., 2022). However, since Tsg101 is also required for phagophore closure (Takahashi et al., 2019), this process is likely unable to dispose of defective endosomes upon Tsg101 knockdown. By contrast, ESCRT-0/Hrs is not required for autophagy in some cell types, such as Drosophila fat body (Rusten et al., 2007). To test if this also applies to motor neurons, we examined GFP-mCherry-Atg8 in Hrs mutant motor neuron cell bodies, and found mCherry-positive, GFP-negative structures, similar to controls (Fig. 2C, E), suggesting that Hrs is not required for autophagic flux in motor neurons. Interestingly, we did observe an increase in the area covered by puncta, indicating that autophagy is induced in Hrs mutants (Fig. 2C). Overall, given that Tsg101 and Hrs have different autophagy phenotypes but similar EV release defects, our results suggest that autophagy and EV traffic are separable functions of ESCRT at the synapse, and that a compensatory (and Tsg101-dependent) autophagy mechanism might be activated to remove defective endosomes in Hrs mutants.
Finally, we further explored whether accumulation of EV cargoes in arrested structures was local to the synapse or occurring throughout the neuron. First, we examined Syt4-GFP levels in motor neuron cell bodies and axons after Tsg101 knockdown, and found that Syt4-GFP accumulated significantly at both locations (Fig. 2F-I). To ask whether the presynaptic accumulations could be due to faster anterograde or slowed retrograde transport of exosome cargo-containing compartments, we next conducted live imaging and kymograph analysis of motor neuron-driven APP-GFP, as well as a mitochondrial marker. We found that Tsg101 knockdown led to a large increase in the number of stationary APP-GFP puncta in axons without affecting the number of compartments undergoing retrograde or anterograde transport (Fig S3A,B), though we observed a small decrease in the retrograde transport rate (Fig S3C). By contrast, we did not observe an increase in the steady state intensity of the mitochondrial marker or see any changes in its transport behavior (Fig S3D-H), suggesting that axonal accumulations are specific to exosome cargo.
Thus, loss of tsg101 leads to accumulation of stationary exosome cargo-containing compartments throughout the neuron, without affecting the transport rates of moving cargoes, suggesting that altered axonal transport kinetics do not underlie synaptic accumulation.
Tsg101 knockdown does not recapitulate all evi or wg phenotypes in synaptic growth or development
Specific depletion of cargo in postsynaptic exosomes (but not the donor presynaptic terminal) upon Tsg101KD provided us with a valuable tool to determine if these cargoes require trans-synaptic transfer for their known synaptic functions. Neuron-derived Wg provides anterograde (to the muscle) and paracrine (to the neuron) signals for NMJ growth, active zone development, and assembly of the postsynaptic apparatus (Miech et al., 2008; Packard et al., 2002). Evi is a multipass transmembrane protein that serves as a carrier for Wg through the secretory system, ultimately leading to its release from the cell, either by conventional exocytosis or via exosomes (Das et al., 2012). At the NMJ, Evi co-transports with Wg into exosomes, and evi mutants phenocopy wg signaling defects, providing support for the hypothesis that Evi/Wg exosomes are required for Wg signaling (Korkut et al. 2009, Koles et al. 2012). However, since Evi is broadly required for many steps of Wg traffic, evi mutants trap Wg in the somatodendritic compartment and prevent its transport into presynaptic terminals (Korkut et al., 2009). Therefore, Wg signaling defects in evi mutants may be due to generalized loss of Wg secretion rather than specific loss of its trans-synaptic transfer. Wg or evi mutants exhibit dramatic reductions in bouton number, together with the appearance of immature boutons with abnormal or missing active zones, fewer mitochondria, aberrant swellings or pockets in the postsynaptic region opposing active zones, and missing areas of PSD95/Discs-Large (DLG)-positive postsynaptic subsynaptic reticulum (Korkut et al., 2009; Packard et al., 2002). In the evi2 mutant, the number of synaptic boutons and the number of active zones (marked by ELKS/CAST/ Bruchpilot (BRP)) were both significantly reduced compared to controls, and the postsynaptic scaffolding molecule DLG frequently exhibited a “feathery” appearance, suggesting defects in postsynaptic assembly (Fig. 3A-E). By contrast, we found that bouton and active zone numbers at Tsg101KD NMJs (which have presynaptic Evi but no Evi exosomes (Fig. 1B)) were not significantly different from controls. Further, active zones in Tsg101KD appeared morphologically normal by TEM (Fig. 2B). We did not observe significant amounts of “feathery” DLG distribution in control or Tsg101KD larvae (2.3% of control NMJs (n=87) and 5.5% of Tsg101KD NMJs (n=87), compared to 51.7% of evi2 mutant NMJs (n=91), p=0.27 for control versus Tsg101KD). We also did not observe significant differences between control and Tsg101KD NMJs in the appearance of subsynaptic reticulum by EM (Fig. 2B) These results indicate that some neuronal Evi and Wg functions are unexpectedly maintained despite loss of detectable postsynaptic Evi exosomes upon Tsg101KD.
In addition to an overall decrease in the number of synaptic boutons, both Wg and evi2 mutants have been reported to show increased numbers of developmentally arrested or “ghost” boutons that feature presynaptic markers such as α-HRP antigens, but lack a postsynaptic apparatus defined by DLG (Korkut et al., 2009). We found that in evi2 mutants, these ghost boutons are more prevalent in anterior segments of the larvae, where overall synaptic growth is more exuberant. Similarly, Tsg101KD animals exhibited a significant increase in ghost boutons in abdominal segment A2 (but not in A3), partially phenocopying the evi2 mutant (Fig, 3 F,G). To further explore this function for other ESCRT components, we quantified ghost boutons upon neuronal ShrubKD. Because it was difficult to recover third instar larvae for this condition, we reared control, evi2, Tsg101KD, and ShrubKD larvae at 20ºC, where RNAi expression from the neuronal GAL4 driver would be weaker. At this temperature, evi2 mutants did not show a ghost bouton phenotype, raising the possibility that this point mutant is only defective at a higher temperature (25ºC). However, both Tsg101KD and ShrubKD exhibited significantly increased ghost bouton number under these conditions. Taken together, these results suggest that some (but not all) aspects of subsynaptic reticulum development may require trans-synaptic transfer of Wg and Evi in exosomes.
Tsg101 knockdown does not recapitulate syt4 phenotypes in activity-dependent structural or functional plasticity
We next explored the functions of the exosome cargo Syt4, which is required for activity-dependent structural and functional plasticity at the NMJ (Barber et al., 2009; Korkut et al., 2013; Piccioli and Littleton, 2014; Yoshihara et al., 2005). Endogenous Syt4 is thought to be generated only by the presynaptic motor neuron, based on the absence of Syt4 transcript in muscle preparations, and the finding that presynaptic RNAi of Syt4 depletes both presynaptic and postsynaptic signals (Korkut 2013). We independently verified that all the Syt4 at the NMJ was derived from the neuron, using a strain in which the endogenous Syt4 locus is tagged at its 3’ end with a switchable TagRFP-T tag, which could be converted to GFP in the genome via tissue-specific GAL4/UAS expression of the Rippase recombinase (Koles et al., 2015; Walsh et al., 2021) (Fig. S4A). Conversion of the tag only in neurons resulted in a bright Syt4-GFP signal both presynaptically and postsynaptically, together with disappearance of the TagRFP-T signal (Fig. S4B). However, conversion of the tag in muscles did not result in any GFP signal, and the TagRFP-T signal remained intact. These results indicate that Syt4 is only expressed in neurons, and support the previous conclusion that the postsynaptic signal is derived from a presynaptically-expressed product (Korkut et al., 2013).
Membrane trafficking mutants such as rab11 and nwk deplete Syt4 from presynaptic terminals (only secondarily reducing its traffic into exosomes), and phenocopy syt4 null mutant plasticity phenotypes (Blanchette et al., 2022; Korkut et al., 2013; Walsh et al., 2021). However, there is no evidence to date that signaling by Syt4 explicitly requires its transfer via exosomes. We first tested the effect of Tsg101KD, which depletes exosomes without diminishing presynaptic Syt4 (Fig. 1A, E), on Syt4-dependent structural plasticity. In this paradigm, spaced high potassium stimulation promotes acute formation of nascent ghost boutons (Ataman et al., 2008; Korkut et al., 2013; Piccioli and Littleton, 2014). These are likely transient structures, and thus are not directly comparable to developmentally arrested ghost boutons such as those that are found in evi mutants (Fernandes et al., 2021). However, to avoid the confounding presence of these immature boutons, we explored the activity-dependent synaptic growth paradigm on muscle 4, where the Tsg101KD animals do not have significantly more ghost boutons than controls under baseline conditions. Unexpectedly, Tsg101KD animals behaved similarly to controls, and exhibited a significant increase in ghost boutons following high K+ spaced stimulation compared to mock stimulation (Fig. 4A-B), suggesting that Syt4 function is preserved in these synapses despite depletion of exosomal Syt4. We were surprised by these results and contacted another laboratory (KPH, BAS) to replicate this experiment independently at muscle 6/7 in segments A3 and A4, and again saw no defect in ghost bouton formation (Fig. S4C-D). KPH next tested the effect of Tsg101KD on Syt4-dependent functional plasticity. In this paradigm, stimulation with 4×100 Hz pulses causes a Syt4-dependent increase in the frequency of miniature excitatory junction potentials (mEJPs), in a phenomenon termed High Frequency-Induced Miniature Release (HFMR) (Korkut et al., 2013; Yoshihara et al., 2005). Tsg101KD animals exhibited similar HFMR to wild type controls, indicating that Syt4 function was not disrupted (Fig. 4C-D). Taken together, our results show that Syt4-dependent structural and functional plasticity at the larval NMJ can occur despite dramatic depletion of exosomal Syt4.
If trans-synaptic transfer of Syt4 in exosomes serves a calcium-responsive signaling function in the muscle, one would expect to find neuronally-derived Syt4 in the muscle cytoplasm. Conversely, if exosomes serve primarily as a proteostatic mechanism to shed neuronal Syt4 for subsequent uptake and degradation in muscles (or glia), then Syt4 would not need to be exposed to the muscle cytoplasm, as it could be taken up in double membrane compartments for degradation via fusion with muscle (or glial) lysosomes. Therefore, we tested whether neuronally-derived Syt4-GFP (for which the GFP moiety is topologically maintained on the cytoplasmic side of membranes in both donor and recipient cells) could be found in the muscle cytoplasm. Using the GAL4/UAS system, we expressed a proteasome-targeted anti-GFP nanobody (deGradFP (Caussinus et al., 2011)) only in neurons or only in muscles. We observed strong depletion of Syt4-GFP fluorescence upon presynaptic deGradFP expression, including a reduction in Syt4 postsynaptic puncta intensity and number, consistent with the presynaptic source of exosomal Syt4-GFP protein (Fig. 4F, H, J). However, we did not observe any difference in either presynaptic or postsynaptic Syt4-GFP levels or puncta number upon deGradFP expression in the muscle (Fig. 4G, I, J), though deGradFP could efficiently deplete DLG as a control postsynaptic protein (Fig. S4E). These results suggest that the majority of postsynaptic Syt4 is not exposed to the muscle cytoplasm.
Discussion
Here we show that the ESCRT pathway is required for EV cargo packaging at the Drosophila larval NMJ, and that these EVs are likely MVE-derived exosomes. We found that ESCRT depletion caused presynaptic accumulation of cargoes, defects in their axonal transport, and a dramatic loss of trans-synaptic transfer in exosomes. Surprisingly, we found that this trans-synaptic transfer is not required for several physiological functions of exosome cargoes Evi and Syt4. Further, neuronally-derived Syt4 could not be detected in the muscle cytoplasm, consistent with findings from Hela cells that the majority of EV cargoes remain in the endosomal system of the recipient cell (O’Brien et al., 2022). Our results suggest that neuronal exosome release for these cargoes serves primarily proteostatic and not signaling functions.
Functions of ESCRT in MVE biogenesis and exosome release at synapses
We found that ESCRT is required for exosome generation and release at the Drosophila larval NMJ. ESCRT components are also required for EV/exosome cargo release from primary neurons in culture (Gong et al., 2016) and Purkinje neurons in vivo (Coulter et al., 2018), but not for exosome release of pathogenic APP variants or Evi from cell lines (Beckett et al., 2013; Cone et al., 2020), underscoring the importance of studying membrane traffic in bona fide neurons. Further, we found that upon ESCRT depletion, cargoes accumulate in intracellular compartments, suggesting that this population of NMJ EVs are MVE-derived exosomes rather than plasma membrane-derived microvesicles. This is consistent with the requirement for endosomal sorting machinery, such as retromer, in their regulation (Walsh et al. 2021).
One major open question is whether exosome-precursor MVEs are generated on-demand in response to local cues at presynaptic terminals, or if they arise in response to global cues and are transported to synapses from other regions of the neuron. Answering this question will require tools to visualize the timecourse of MVE biogenesis in neurons, as have been developed in cultured non-neuronal cells (Wenzel et al., 2018). In addition, future methods (e.g. optogenetic) for acute and localized inhibition of ESCRT will reveal whether arrested structures first appear locally at the synapse and are only later transported into axons and cell bodies, and/or if they are generated far from the site of release at the synapse. These experiments will be critical for understanding when and where local or global signaling events impinge on exosome biogenesis. Interestingly, activity-dependent delivery of Hrs to presynaptic terminals is critical for proteostasis of synaptic vesicle proteins (Birdsall et al., 2022; Boecker and Holzbaur, 2019; Sheehan et al., 2016). If MVEs are generated on-demand at synapses, Hrs transport could similarly underlie the activity-dependence of exosome release, which has been reported in many (but not all) neuronal experimental systems, and remains poorly understood (Ataman et al., 2008; Fauré et al., 2006; Lachenal et al., 2011; Lee et al., 2018; Vilcaes et al., 2021).
Other synaptic functions of ESCRT
ESCRT is best-known for its functions in MVE biogenesis, but has many other potential synaptic roles including in autophagy, lipid transfer and membrane repair (Vietri et al., 2020). Our results show that the function of ESCRT in exosome release is likely separate from its roles in autophagy, since several canonical autophagy mutants do not phenocopy the exosome trafficking defects seen upon ESCRT depletion, and Hrs mutants exhibit exosome but not autophagic flux defects. Interestingly, we found that atg mutants led to an overall reduction in levels of the exosome cargo Nrg, raising the possibility that other degradative pathways are upregulated at synapses when autophagy is blocked. Tsg101 is also involved in lipid transfer to mitochondria (Wang et al., 2021), but we did not detect obvious defects in mitochondria in motor neuron axons, as were seen in Tsg101-mutant Drosophila adult wing sensory neurons (Lin et al., 2021). Thus, while ESCRT has many cellular activities, our experiments separate these functions to specifically narrow down its role in neuronal exosome release.
Our data also raise the possibility of a novel synaptic proteostasis mechanism which might be termed “endosomophagy”, as has been seen in cell culture (Millarte et al., 2022; Wang et al., 2022; Zellner et al., 2021), and adds to the numerous intersections between endolysosomal traffic and autophagy in neurons (Boecker and Holzbaur, 2019). Many organelles are selectively targeted for macroautophagy via compartment-specific receptors (Lamark and Johansen, 2021), but such a process has not been specifically described for neuronal endosomes/MVEs. Our results suggest that autophagy is induced in ESCRT mutant synapses, presumably to dispose of aberrant endosomes, with different outcomes in Tsg101KD versus Hrs mutants. Tsg101KD led to aberrant autophagic vacuoles and reduced autophagic flux, perhaps due to a secondary role for ESCRT-1/Tsg101 in phagophore closure (Takahashi et al., 2019) or another step of autophagy. By contrast we found that Hrs mutants do not show these structures, either by light microscopy of the autophagic flux reporter GFP-mCherry-ATG8, or in previously published TEM of the NMJ (Lloyd et al., 2002). Instead, Hrs mutants exhibit induction of autophagy but normal autophagic flux in motor neurons, together with a moderate reduction in exosome cargo levels. Together, these results suggest that aberrant exosome-cargo-containing MVEs may be removed from Hrs mutant synapses by a compensatory, Tsg101-dependent autophagy pathway.
Implications for the physiological functions of exosomes
The majority of functional studies of EVs involve isolating EV subpopulations (at various degrees of homogeneity) from cell culture supernatants, applying them to target cells or tissues, and assessing their biological effects (Théry et al., 2018). Additional mechanistic insight has been obtained by eliminating specific cargo molecules from the donor cells before EV isolation, to determine if these molecules are required for EV bioactivity. While these approaches are very useful for determining therapeutic uses for EVs, they have several major limitations for understanding their normal functions in vivo. First, it is difficult to determine the concentration of EVs that a target cell would normally encounter, in order to design a physiologically relevant experiment. Second, while these types of experiments inform what EVs can do, they do not show that EV transfer is necessary for that signaling function in vivo. Removing the signaling cargo from the donor cell also does not show the necessity of EV transfer for biological functions, since the cargo could be acting cell autonomously in the donor cell, or could signal to a neighboring cell by another trafficking route. Indeed, previous studies at the Drosophila larval NMJ, which has been an important model system for the in vivo functions of EV traffic, have conducted tests for EV cargo activity in evi or rab11 mutants, though we and others have shown that this results in depletion of cargo from the presynaptic donor cell in addition to loss of EVs (Ashley et al., 2018; Blanchette et al., 2022; Korkut et al., 2013, 2009; Walsh et al., 2021). Ultimately, determining if transfer of a cargo in EVs is necessary for its signaling function requires blocking EV transfer specifically, which we were able to achieve at ESCRT-depleted synapses.
Neuronally derived Wg is required and sufficient for synaptic growth, and is required together with glia-derived Wg to organize postsynaptic glutamate receptor fields (Kerr et al., 2014; Miech et al., 2008; Packard et al., 2002). Therefore, if transsynaptic transfer of Evi was required for Wg signaling, we would have expected to see a dramatic reduction in synaptic growth at ESCRT-depleted synapses, as well as disruptions in postsynaptic development and organization (Korkut et al., 2009; Miech et al., 2008; Packard et al., 2002). Instead, we observed no significant change in bouton or active zone number relative to controls. wg-phenocopying defects in postsynaptic development were also not observed by electron microscopy in CHMPIIBintron5 (West et al., 2015) or Hrs-mutant synapses (Lloyd et al., 2002), or in our data from ESCRT mutants. Similarly, Hsp90 mutants attenuate Evi exosome release by disrupting MVE-plasma membrane fusion, but do not result in a strong disruption of active zone or postsynaptic development (Lauwers et al., 2018). Thus, Evi release in exosomes does not correlate with effective Wg signaling. Importantly, it is likely that Hsp90 and ESCRT mutant synapses do secrete Wg, albeit by conventional secretion rather than an exosomal mechanism (Beckett et al., 2013), and that the primary function for Evi is to traffic Wg to the presynaptic terminal and maintain its levels there. However, we did find that knockdown of the ESCRT components Tsg101 and Shrub recapitulated one phenotype of evi and Wg mutants, which is an increase in developmentally arrested ghost boutons. This suggests that transynaptic transfer of Evi and Wg may be required for some aspects of assembly of the post-synaptic apparatus during larval development.
Syt4 protein is thought to act in the postsynaptic muscle (Adolfsen et al., 2004; Barber et al., 2009; Harris et al., 2016), but its endogenous transcript is not expressed in this tissue, leading to the prevailing model of transynaptic transfer from the presynaptic neuron in exosomes (Korkut et al., 2013). However, our results show that transynaptic transfer in exosomes can be blocked without affecting the signaling activities of Syt4, and that the majority of postsynaptic Syt4 is not exposed to the muscle cytoplasm. The main evidence for a muscle requirement for Syt4 is that re-expression of Syt4 using muscle-specific GAL4 drivers is sufficient to rescue structural and functional plasticity defects of the syt4 null mutant (Korkut et al., 2013; Piccioli and Littleton, 2014; Yoshihara et al., 2005). This is difficult to reconcile with our findings that Tsg101KD animals lack detectable postsynaptic Syt4, but do not phenocopy syt4 mutants. There are several possible explanations for this conundrum. First, we cannot completely rule out the possibility that small amounts of residual exosomal Syt4 after Tsg101 knockdown (perhaps transferred by a non-exosomal pathway, and distributed diffusely in the muscle) are sufficient to drive a transynaptic signal. An argument against this possibility is that nwk and rab11 mutants also have trace amounts of Syt4 postsynaptically, and do strongly phenocopy the syt4 null mutant (presumably since they also deplete Syt4 from the presynaptic compartment) (Korkut et al., 2013; Walsh et al., 2021; Blanchette et al., 2022). Second, it is possible that the muscle GAL4 drivers used in these previous rescue studies have some leaky expression in the neuron. Third, ectopically muscle-expressed Syt4 might have a neomorphic function in the muscle that bypasses the loss of neuronal Syt4, or else it could be retrogradely transported to the neuron. Indeed, muscle-expressed Syt4 is localized in close apposition to the presynaptic membrane (Harris et al., 2016).
Conclusion
Why are Evi and Syt4 trafficked into exosomes, if not for a signaling function? Local exosome release could serve as a proteostatic mechanism for synapse-specific control of signaling cargo levels. Indeed, the amount of cargo loaded into exosomes could be tuned by regulating endosomal sorting via retromer (Walsh et al., 2021), or by controlling the rate of endocytic flux into the Rab11-dependent recycling pathway (Blanchette et al., 2022). Through these mechanisms, neurons might achieve local control of synaptogenic or plasticity-inducing signaling pathways, in a much more rapid and spatially directed fashion than transcriptional or translational regulation. Importantly, our results do not rule out signaling functions for these cargoes in other contexts or neuronal cell types, or for other EV cargoes. Indeed, extensive evidence supports signaling functions for neuronal EVs in multiple contexts (Gassama and Favereaux, 2021; Lizarraga-Valderrama and Sheridan, 2021; Schnatz et al., 2021). However, our data encourage future hypothesis-challenging experiments for EV functions using membrane trafficking mutants that disrupt EV release specifically.
Materials and Methods
Drosophila culture
Flies were cultured using standard media and techniques. Flies used for experiments were maintained at 25°C, except for experiments using Shrub-RNAi, which were maintained at 20°C. For detailed information on fly stocks used, see Table S1, and for detailed genotype information for each figure panel, see Table S3.
Immunohistochemistry
Wandering 3rd instar larvae were dissected in HL3.1 and fixed in HL3.1 with 4% paraformaldehyde for 45 minutes. Washes and antibody dilutions were conducted using PBS containing 0.2% Triton X-100 (0.2% PBX). Primary antibody incubations were conducted overnight at 4°C, and secondary antibody incubations for 1-2 hours at room temperature. α-HRP incubations were conducted either overnight at 4°C or for 1-2 hours at room temperature. Prior to imaging, fillets were mounted on slides with Vectashield (Vector Labs). For detailed information on antibodies used in this study, see Table S2.
Activity-induced synaptic growth
High K+ spaced stimulation was performed as described (Piccioli and Littleton, 2014). Briefly, 3rd instar larvae were dissected in HL3 solution at room temperature (in mM, 70 NaCl2, 5 KCl, 0.2 CaCl2, 20 MgCl2, 10 NaHCO3, 5 trehalose, 115 sucrose, and 5 HEPES (pH=7.2)). Dissecting pins were then moved inward to relax the fillet to 60% of its original size, and then stimulated 3 times in high K+ solution (in mM, 40 NaCl2, 90 KCl, 1.5 CaCl2, 20 MgCl2, 10 NaHCO3, 5 trehalose, 5 sucrose, and 5 HEPES (pH=7.2)) for 2 minutes each, with 10-minute HL3 incubations in between stimulation while on a shaker at room temperature. Following the 3rd and final stimulation, larvae were incubated in HL3 (approximately 2 minutes) and stretched to their initial length. Mock stimulations were performed identically to the high K+ stimulation assay, except HL3 solution was used in place of high K+ solution. Larvae were then fixed in 4% PFA in HL3 solution for 15 minutes and then stained and mounted as above.
Electrophysiology
Wandering 3rd instar larvae were dissected in HL3 saline (Stewart et al., 1994). Recordings were taken using an AxoClamp 2B amplifier (Axon Instruments, Burlingame, CA). A recording electrode was filled with 3M KCl and inserted into muscle 6 at abdominal segments A3 or A4. A stimulating electrode filled with saline was used to stimulate the severed segmental nerve using an isolated pulse stimulator (2100; A-M Systems). HFMR was induced by four trains of 100 Hz stimuli spaced 2 s apart in 0.3 mM extracellular Ca2+. Miniature excitatory junctional potentials (minis) were recorded 2 min before and 10 min after HFMR induction. Mini frequency at indicated time points was calculated in 10-s bins. Fold enhancement was calculated by normalizing to the baseline mini frequency recorded prior to HFMR induction. Analyses were performed using Clampfit 10.0 software (Molecular Devices, Sunnyvale, CA). Each n value represents a single muscle recording, with data generated from at least six individual larvae of each genotype arising from at least two independent crosses. Resting membrane potentials were between -50 mV and -75 mV and were not different between genotypes. Input resistances were between 5 MΩ and 10 MΩ and were not different between genotypes.
Electron microscopy
Wandering 3rd instar larvae were dissected and fixed in 1% glutaraldehyde and 4% paraformaldehyde in 1% (0.1M) sodium cacodylate buffer overnight at 4°C. Samples were postfixed in 1% osmium tetroxide and 1.5% potassium ferrocyanide for 1 h, then 1% aqueous uranyl acetate for 0.5 h. Stepwise dehydration was conducted for 10 min each in 30%, 50%, 70%, 85%, and 95% ethanol, followed by 2× 10 min in 100% ethanol. Samples were transferred to 100% propylene oxide for 1 h, then 3:1 propylene oxide and 812 TAAB Epon Resin (epon, TAAB Laboratories Equipment Ltd.) for 1 h, then 1:1 propylene oxide:epon for 1 h and then left overnight in a 1:3 mixture of propylene oxide:epon. Samples were then transferred to fresh epon for 2 h. Samples were then flat-embedded and polymerized at 60°C for 48 h, and remounted for sectioning. 70-µm-thin sections were cut on a Leica UC6 Ultramicrotome (Leica Microsystems), collected onto 2×1 mm slot grids coated with formvar and carbon, and then poststained with lead citrate (Venable and Coggeshall, 1965). Grids were imaged using a FEI Morgagni transmission electron microscope (FEI) operating at 80 kV and equipped with an AMT Nanosprint5 camera.
Imaging and quantification
Acquisition
Analysis of EV cargoes and bouton morphology were conducted at muscle 6/7 NMJ from segments A2 and A3, or for Fig. 4A at muscle 4 from segments A2-A4. Z-stacks were acquired using a Nikon Ni-E upright microscope equipped with a Yokogawa CSU-W1 spinning disk head, an Andor iXon 897U EMCCD camera, and Nikon Elements AR software. A 60X (n.a. 1.4) oil immersion objective was used to image NMJs, cell bodies, and fixed axons. Data in Fig. S4C were acquired with a Zeiss LSM 800 confocal microscope using a 40x (n.a. 1.4) oil immersion objective and Zen Black 2.3 software. For colocalization and puncta analysis branches from muscle 6/7 NMJ from segments A2 and A3 were taken using Zen Blue software on a Zeiss LSM880 Fast Airyscan microscope in super resolution acquisition mode, using a 63X (n.a. 1.4) oil immersion objective. For axon transport, timelapse images were taken on the same Nikon Ni-E microscope described above. Images were taken of axon bundles proximal to the ventral ganglion (roughly within 100-300 µm). For APP transport, timelapse images were acquired for 3 minutes using 60X (n.a. 1.4) oil immersion objective. For mitochondria timelapse, images were acquired for 7 minutes using 60X (n.a. 1.0) water immersion objective. 9 Z slices were collected per frame (Step size 0.3 µm, with no acquisition delay between timepoints, resulting in a frame rate of 2.34-2.37 sec/frame). To visualize moving particles for mitochondria, a third of the axon in the field of view was photobleached using an Andor Mosaic digital micromirror device operated by Andor IQ software, to eliminate fluorescence from stationary particles that would interfere with visualization of particles moving into the bleached region. Image acquisition settings were identical for all images in each independent experiment.
EV quantification and colocalization
Volumetric analysis was performed using Volocity 6.0 software. For each image, both type 1s and 1b boutons were retained for analysis while axons were cropped out. The presynaptic volume was defined by an HRP threshold, excluding objects smaller than 7 µm3 and closing holes. The postsynaptic region was defined by a 3 µm dilation of the HRP mask. However, for Evi-GFP, where the presynaptic signal vastly exceeded postsynaptic signal, we analyzed only the distal 2 µm of this postsynaptic dilation region to eliminate the bleed-over haze from the presynaptic signal. EV cargo and Rab signals were manually thresholded to select particles brighter than the muscle background. EV cargo integrated density in these thresholded puncta was normalized to the overall presynaptic volume. These values were further normalized to the mean of the control to produce a “normalized puncta intensity” value for each NMJ. For colocalization, the overlap of the two channels was measured in Volocity 6.0 and used for calculation of Mander’s coefficients.
Quantification of electron micrographs
A single medial section of each bouton was selected for analysis. Two experimenters, blinded to genotype, together recorded the presence of autophagic vacuoles, including phagopores (double or dense membrane but not closed; note that depending on the plane of section, some of these may appear as autophagosomes), autophagosomes (contents with similar properties to the cytoplasm, fully enclosed in the section by a double membrane), and autolyososomes (contents are electron dense) (Lucocq and Hacker, 2013; Nagy et al., 2015). We also evaluated whether boutons lacked SSR, or featured postsynaptic pockets (electron-lucent areas extending at least 300 nm from the presynaptic membrane (Packard et al., 2002).
Quantification of GFP-mCherry-Atg8 distribution
A single field of view confocal stack (62×62 µm) from the larval ventral ganglion, containing 10-15 Vglut-expressing cell bodies, was manually thresholded in Volocity 6.0 software to segment and measure the volume and integrated fluorescence density of soma, GFP puncta, and mCherry puncta. The overlap between the GFP and mCherry channels was used for the calculation of the Mander’s coefficient (fraction of total mCherry-puncta integrated density found in the GFP-puncta positive volume).
Axon and cell body measurements
To measure intensity of EV cargoes in axons, axons proximal to the ventral ganglion (within 100-300 µm) were imaged as described above. Images were analyzed in Fiji by making sum projections, cropping out unwanted debris or other tissue and making a mask on the HRP. The total intensity of the EV cargo was measured within the masked HRP area. For cell bodies, EV cargo intensity was measured from a middle slice through the motor neuron cell body layer of the ventral ganglion using Fiji.
Quantification of live axonal trafficking of APP-GFP and Mito-GFP puncta
To quantify APP-GFP and mitochondria dynamics in live axons, maximum intensity projections of time course images were processed in Fiji to subtract background and adjust for XY drift using the StackReg plugin. Kymographs were generated from 1-4 axons per animal using the Fiji plugin KymographBuilder. Kymographs were blinded and number of tracks were manually counted. The minimum track length measured was 3 μm with most tracks above 5 μm. Velocity was measured by calculating the slope of the identified tracks.
Bouton quantification
The experimenter was blinded to genotypes and then manually counted the total number of type 1 synaptic boutons on the NMJ on muscle 6 and 7 in the abdominal segments A2 and A3 of third instar wandering larvae. A synaptic bouton was considered each spherical varicosity, defined by the presence of the synaptic vesicle marker Synaptotagmin 1, the active zone marker Bruchpilot (Brp) and the neuronal membrane marker Hrp. For quantifying ghost boutons (basal and activity-induced), the experimenter was blinded to genotype and condition and ghost boutons were quantified as α-HRP-positive structures with a visible connection to the main NMJ arbor, and without α-DLG staining. For quantifying DLG “featheriness”, the experimenter was blinded to genotype and scored the number of NMJs with at least one region of fenestrated Dlg that extended far from the bouton periphery.
Active zone quantification
To count the active zones in fluorescence micrographs, Brp-stained punctae were assessed on maximum intensity projection images. The Trainable Weka Segmentation (TWS) machine-learning tool (https://doi.org/10.1093/bioinformatics/btx180) in Fiji was used to manually annotate Brp-positive punctae with different fluorescence intensities, and to train a classifier that automatically segmented the Brppositive active zones. The objects segmented via the applied classifier were subjected to Huang auto thresholding to obtain binary masks. Next, we applied a Watershed processing on the binary image, to improve the isolation of individual neighboring active zones from the diffraction limited images. We performed particle analysis on the segmented active zones and obtained their number, area, and integrated density. The number of active zones was normalized to the NMJ area. To determine the NMJ area using TWS, we trained the classifier by annotating the HRP positive NMJ on maximum intensity projections of the HRP channel. Axons were manually cropped from the image before TWS. The segmented HRP area was subjected to Huang auto thresholding, the binary masks were selected and the NMJ area was obtained via the “Analyze particle” function in FIJI of particles larger than 5 µm (to eliminate from the analysis residual HRP EV debris segmented in a very few images).
Statistics
All statistical measurements were performed in GraphPad Prism 6 (see Table S3). Comparisons were made separately for presynaptic and postsynaptic datasets, due to differences between these compartments for intensity, signal-to-noise ratio, and variance. Datasets were tested for normality, and statistical significance was tested using unpaired two-tailed Student’s t tests or Mann–Whitney tests (if number of conditions was two) or ANOVA followed by Tukey’s tests or Kruskal–Wallis followed by Dunn’s test (if number of conditions was greater than two). For categorical measurements of autophagic vacuoles and DLG distribution, Fisher’s exact test was used. Statistical significance is indicated as *P < 0.05; **P < 0.01; ***P < 0.001.
Author contributions
ECD, KPH, KK, MFP, BAS, AAR designed the study and experiments. ECD, KPH, KK,, MFP, MR, RCS conducted the experiments. ECD, KPH, KK, MFP, BE, MR, RCS and AAR performed the analyses. ECD and AAR wrote the manuscript, and all authors edited the manuscript. This article contains supporting information (4 Supplemental Figures and 3 tables).
Competing interest statement
The authors declare no competing financial interests.
Acknowledgements
We thank the Developmental Studies Hybridoma Bank created by the NICHD of the NIH, and the Bloomington Drosophila Stock Center (Indiana University, Bloomington, IN, NIH P40OD018537). We thank Berith Isaac and the Brandeis Electron Microscopy Facility for assistance with EM, Michael Boutros, Josie Clowney, and Tor Erik Ruston for fly lines, Harald Stenmark for discussion of endosomophagy, and Cassie Blanchette and Steve Del Signore for comments on the manuscript. This work was supported by NINDS grants R01 NS103967 to A.A.R. and F32 NS120909 to E.C.D.