Eating Locally: Microautophagy and Protein Turnover at the Synapse
Authors: Peter S. McPherson
Journal: Neuron (2015)

Mechanisms of local protein quality control in the presynaptic nerve terminal remain largely unknown. In this issue of Neuron, Uytterhoeven et al., 2015Uytterhoeven V. Lauwers E. Maes I. Miskiewicz K. Melo M.N. Swerts J. Kuenen S. Wittocx R. Corthout N. Marrink S.-J. et al.Neuron. 2015; 88 (this issue): 735-748Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar describe a form of synaptic endosomal microautophagy controlling protein rejuvenation, critical for optimal synaptic activity. Mechanisms of local protein quality control in the presynaptic nerve terminal remain largely unknown. In this issue of Neuron, Uytterhoeven et al., 2015Uytterhoeven V. Lauwers E. Maes I. Miskiewicz K. Melo M.N. Swerts J. Kuenen S. Wittocx R. Corthout N. Marrink S.-J. et al.Neuron. 2015; 88 (this issue): 735-748Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar describe a form of synaptic endosomal microautophagy controlling protein rejuvenation, critical for optimal synaptic activity. The main biosynthetic and protein quality control machineries of a neuron are located in the cell body. The axon that emanates from the cell body can transverse vast distances, up to a meter in the case of a human spinal motor neuron (an unimaginable 30 m for a blue whale), before making a synaptic contact on a target tissue. Thus, the presynaptic compartment must function somewhat independently of the cell body. The most striking example of this autonomous function is seen with the exo/endocytic recycling of synaptic vesicles, wherein vesicles undergo continuous rounds of exocytic fusion and neurotransmitter release followed by local reformation and neurotransmitter loading (Kononenko and Haucke, 2015Kononenko N.L. Haucke V. Neuron. 2015; 85: 484-496Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The synaptic vesicle cycle is the primary source of activity-driven metabolic need in the nervous system, and this massive demand is fed by local ATP production (Rangaraju et al., 2014Rangaraju V. Calloway N. Ryan T.A. Cell. 2014; 156: 825-835Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). With these robust metabolic and membrane trafficking activities comes a requirement for local protein quality control, as proteins are subjected to wear-and-tear including oxidative stress, and yet the mechanisms that maintain quality control of synaptic proteins remain largely unknown. Eukaryotic cells have evolved multiple mechanisms to turnover non-functional proteins, including proteosomal degradation of soluble proteins, endosomal/lysosomal sorting of transmembrane proteins, and autophagy. There are three main types of autophagy, which likely co-exist in all mammalian cells: (1) macroautophagy, where portions of cytosol or entire organelles are engulfed by a double membrane structure before transport to and fusion with lysosomes; (2) microautophagy, where cytosolic proteins are sequestered directly by the lysosomal membrane; and (3) chaperone-mediated autophagy, where cytosolic proteins with a specific tag are recognized by the heat shock cognate protein 70 (Hsc70), which targets them to the surface of lysosomes for translocation through a protein pore to the lysosomal lumen (Cuervo, 2010Cuervo A.M. Trends Endocrinol. Metab. 2010; 21: 142-150Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). More recently, a unique form of microautophagy called endosomal microautophagy has been described. Specific cytosolic proteins (marked with the same tag as those undergoing chaperone-mediated autophagy) are recognized by Hsc70, which targets them to the surface of late endosomes, where they undergo inward invagination for delivery to intraluminal vesicles of a multivesicular body, with eventual degradation in lysosomes (Sahu et al., 2011Sahu R. Kaushik S. Clement C.C. Cannizzo E.S. Scharf B. Follenzi A. Potolicchio I. Nieves E. Cuervo A.M. Santambrogio L. Dev. Cell. 2011; 20: 131-139Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar). Autophagy pathways control numerous cellular functions and have thus received extensive study, and yet it remains unclear whether any of these autophagic mechanisms function at synapses. In this issue of Neuron, Verstreken and colleagues demonstrate endosomal microautophagy activity in Drosophila synapses and provide insight into its mechanism of function and regulation (Uytterhoeven et al., 2015Uytterhoeven V. Lauwers E. Maes I. Miskiewicz K. Melo M.N. Swerts J. Kuenen S. Wittocx R. Corthout N. Marrink S.-J. et al.Neuron. 2015; 88 (this issue): 735-748Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Following from a rationale that most autophagic processes require membrane remodeling, Uytterhoeven et al., 2015Uytterhoeven V. Lauwers E. Maes I. Miskiewicz K. Melo M.N. Swerts J. Kuenen S. Wittocx R. Corthout N. Marrink S.-J. et al.Neuron. 2015; 88 (this issue): 735-748Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar devised an in vitro screen, in which a library of neuron-enriched Drosophila proteins, expressed in bacteria, were tested for their ability to deform fluorescently labeled membrane vesicles. Among the numerous proteins identified, including several with defined membrane-deforming activities, was Hsc70-4, which is 87% identical to mammalian Hsc70 and conserves all key domains. In addition to its roles in autophagy, Hsc70 facilitates the proper folding of newly translated or misfolded proteins (Liu et al., 2012Liu T. Daniels C.K. Cao S. Pharmacol. Ther. 2012; 136: 354-374Crossref PubMed Scopus (180) Google Scholar), yet a role for Hsc70 in membrane deformation had not been reported. Uytterhoeven et al., 2015Uytterhoeven V. Lauwers E. Maes I. Miskiewicz K. Melo M.N. Swerts J. Kuenen S. Wittocx R. Corthout N. Marrink S.-J. et al.Neuron. 2015; 88 (this issue): 735-748Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar confirmed Hsc70-4-mediated membrane tubulation in vitro and demonstrated that it requires Hsc70 oligomerization. Moreover, expression of Hsc70-4 in yeast leads to enhanced formation of intraluminal tubules and vesicles in the vacuole (equivalent of a mammalian lysosome) supporting that the membrane deforming activity also functions in vivo. Hsc70 is composed of an N-terminal ATPase domain, followed by a binding domain for client substrates and a C-terminal domain that contains a polybasic stretch required for electrostatic membrane interactions. The authors generated a series of mutant constructs including Hsc70D10N, which disrupts ATPase activity, and Hsc703KA, which converts evolutionarily conserved lysines 531, 533, and 539 to alanine, disrupting membrane interactions. Interestingly, Hsc70D10N, which is devoid of chaperone activity, retains its ability to tubulate membranes both in vitro and in vivo, whereas Hsc703KA fails to tubulate membranes but has normal chaperone activity. Thus, oligomerized Hsc70-4 causes membrane deformation dependent on membrane interactions but independent of chaperone activity. Hsc70 has an established role in endosomal microautophagy, a process requiring membrane deformation (Sahu et al., 2011Sahu R. Kaushik S. Clement C.C. Cannizzo E.S. Scharf B. Follenzi A. Potolicchio I. Nieves E. Cuervo A.M. Santambrogio L. Dev. Cell. 2011; 20: 131-139Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar), and the protein is abundant in the synapse (Wilhelm et al., 2014Wilhelm B.G. Mandad S. Truckenbrodt S. Kröhnert K. Schäfer C. Rammner B. Koo S.J. Claßen G.A. Krauss M. Haucke V. et al.Science. 2014; 344: 1023-1028Crossref PubMed Scopus (466) Google Scholar). It therefore seemed likely that Hsc70 participates in endosomal microautophagy in the synapse but assays to test this hypothesis were lacking. Cytosolic proteins targeted for endosomal microautophagy contain one or more copies of a pentapeptide motif biochemically related to the sequence KFERQ (Sahu et al., 2011Sahu R. Kaushik S. Clement C.C. Cannizzo E.S. Scharf B. Follenzi A. Potolicchio I. Nieves E. Cuervo A.M. Santambrogio L. Dev. Cell. 2011; 20: 131-139Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar), originally described for guiding cytosolic proteins to lysosomes for degradation (Dice, 1990Dice J.F. Trends Biochem. Sci. 1990; 15: 305-309Abstract Full Text PDF PubMed Scopus (529) Google Scholar). Uytterhoeven et al., 2015Uytterhoeven V. Lauwers E. Maes I. Miskiewicz K. Melo M.N. Swerts J. Kuenen S. Wittocx R. Corthout N. Marrink S.-J. et al.Neuron. 2015; 88 (this issue): 735-748Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar generated flies expressing the N- or C-terminal portions of a fluorescent protein fused to KFERQ, reasoning that fluorescence would occur when the two moieties are concentrated following targeting for microautophagy. Focusing on the neuromuscular junction (NMJ), they observe that when expressed together, the constructs give a fluorescent signal in synaptic boutons, which is enhanced by overexpression of Hsc70. Remarkably, the signal is also increased in a fly line with loss of function of small glutamate-rich teratricopeptide repeat-containing protein (Sgt). Sgt is a co-chaperone for Hsc70 that promotes ATPase activity and chaperone function (Tobaben et al., 2001Tobaben S. Thakur P. Fernández-Chacón R. Südhof T.C. Rettig J. Stahl B. Neuron. 2001; 31: 987-999Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Interesting, Sgt inhibits Hsc70 oligomerization and Hsc70-mediated membrane tubulation, consistent with its inhibition of the fluorescence-based synaptic microautophagy assay (Uytterhoeven et al., 2015Uytterhoeven V. Lauwers E. Maes I. Miskiewicz K. Melo M.N. Swerts J. Kuenen S. Wittocx R. Corthout N. Marrink S.-J. et al.Neuron. 2015; 88 (this issue): 735-748Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Taken together, these data lead to a model in which Hsc70 oligomerization deforms membranes contributing to the inward invagination of intraluminal vesicles required for synaptic endosomal microautophagy (Figure 1). Interaction of Hsc70 with Sgt switches the function of Hsc70 away from this activity and toward chaperone-mediated protein folding. In this way, Hsc70 controls synaptic protein quality control in two ways, by refolding proteins together with Sgt or, if this fails, targeting proteins to endosomal microautophagy. The authors next sought to test the function of Hsc70 in endosomal microautophagy in vivo. Using transmission electron microscopy, they find that overexpression of wild-type Hsc70 or disruption of Sgt enhances the formation of synaptic multivesicular structures. They also explored the turnover of synaptic proteins that contain microautophagy recognition motifs. Disruption of the function of Hsc70 or Sgt enhances or reduces, respectively, the levels of four different synaptic proteins containing one or more motifs but has no influence on additional synaptic proteins lacking such motifs. For one such protein, comatose (known in mammals as NSF), mutation of its two microautophagy recognition motifs renders it insensitive to changes in Hsc70 or Sgt function. Since microautophagy stimulates degradation of synaptic proteins, Uytterhoeven et al., 2015Uytterhoeven V. Lauwers E. Maes I. Miskiewicz K. Melo M.N. Swerts J. Kuenen S. Wittocx R. Corthout N. Marrink S.-J. et al.Neuron. 2015; 88 (this issue): 735-748Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar hypothesized that the net influence of stimulating this process is generation of a younger synaptic protein pool. This was assessed using a variant of mCherry fluorescent that converts from blue to red fluorescence over time (Subach et al., 2009Subach F.V. Subach O.M. Gundorov I.S. Morozova K.S. Piatkevich K.D. Cuervo A.M. Verkhusha V.V. Nat. Chem. Biol. 2009; 5: 118-126Crossref PubMed Scopus (119) Google Scholar). A transgenic line containing this fluorescent timer protein fused to comatose was crossed with various Hsc70 lines, control or mutants, revealing that expression of Hsc70 constructs that enhance endosomal microautophagy lead to more blue fluorescence, indicating a younger pool of comatose protein. Finally, using two-electrode voltage clamp at NMJs, Uytterhoeven et al., 2015Uytterhoeven V. Lauwers E. Maes I. Miskiewicz K. Melo M.N. Swerts J. Kuenen S. Wittocx R. Corthout N. Marrink S.-J. et al.Neuron. 2015; 88 (this issue): 735-748Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar discovered that overexpression of Hsc70-4 or disruption of Sgt function leads to enhanced mean excitatory junctional current (EJC) amplitude. There is no change in miniature EJC amplitude or in release probability of a vesicle, but instead the enhanced neurotransmitter release results from an increase in the size of the readily releasable pool of synaptic vesicles. Thus, stimulating endosomal microautophagy at the synapse by releasing Sgt-mediated inhibition of Hsc70 leads to increased turnover of synaptic proteins, a younger and presumably more functional pool of synaptic proteins, and an increased pool of readily releasable vesicles allowing for more robust neurotransmitter release. The study leaves several outstanding questions, notably, what causes the release of Sgt-mediated inhibition of Hsc70, triggering the switch from chaperone function to endosomal microautophagy function? A complex of SNARE proteins that includes the plasma membrane t-SNARE SNAP-25 drives the exocytic fusion of synaptic vesicles (Kononenko and Haucke, 2015Kononenko N.L. Haucke V. Neuron. 2015; 85: 484-496Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The conformational quality control of SNAP-25 is indispensible for the nerve terminal to fire at high frequency and abnormal conformers of SNAP-25 accumulate in an activity-dependent manner, causing defective SNARE complex assembly and neurodegeneration (Sharma et al., 2011Sharma M. Burré J. Südhof T.C. Nat. Cell Biol. 2011; 13: 30-39Crossref PubMed Scopus (181) Google Scholar). It is thus conceivable that reactive intermediates produced during the SNARE cycle could induce a general age-dependent neurotoxicity (Mattson, 2003Mattson M.P. Neuromolecular Med. 2003; 3: 65-94Crossref PubMed Scopus (413) Google Scholar). Hsc70/Sgt form a trimeric complex with the synaptic vesicle-associated co-chaperone cysteine string protein α (Tobaben et al., 2001Tobaben S. Thakur P. Fernández-Chacón R. Südhof T.C. Rettig J. Stahl B. Neuron. 2001; 31: 987-999Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and this complex uses the chaperone activity of Hsc70 to refold fusion incompetent SNAP-25 conformers (Sharma et al., 2011Sharma M. Burré J. Südhof T.C. Nat. Cell Biol. 2011; 13: 30-39Crossref PubMed Scopus (181) Google Scholar). Intriguingly, both Hsc70 and Sgt are upregulated with enhanced synaptic activity (Sharma et al., 2011Sharma M. Burré J. Südhof T.C. Nat. Cell Biol. 2011; 13: 30-39Crossref PubMed Scopus (181) Google Scholar). It is thus conceivable that changes in the relative levels of the two proteins in response to specific patterns of synaptic activity switch Hsc70 between its refolding chaperone function and its endosomal microautophagy function. As such, Hsc70 would switch from trying to refold SNAP-25 to targeting the protein, which has two microautophagy recognition motifs, to endosomal microautophagy. Moreover, Uytterhoeven et al., 2015Uytterhoeven V. Lauwers E. Maes I. Miskiewicz K. Melo M.N. Swerts J. Kuenen S. Wittocx R. Corthout N. Marrink S.-J. et al.Neuron. 2015; 88 (this issue): 735-748Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar find that ATP prevents oligomerization and the membrane deformation properties of Hsc70. Thus, in the presence of abundant ATP, Hsc70 would function as a chaperone but, with ATP deficiency, it would switch to a protein degradation mode. Remarkably, despite the fact that ATP synthesis is driven locally in the synapse and there is a large reservoir of ATP, the incredible metabolic demands of the synapse means that even brief interruption in activity-stimulated ATP synthesis impairs presynaptic function (Rangaraju et al., 2014Rangaraju V. Calloway N. Ryan T.A. Cell. 2014; 156: 825-835Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). Thus, a simple switch in the local availability of ATP could provide an attractive mechanism to balance refolding versus degradation of synaptic proteins. It is likely that synaptic endosomal microautophagy is but one of many processes that will be discovered to function in the local environment of the synapse to ensure the controlled activity of this key functional unit of the nervous system. Hsc70-4 Deforms Membranes to Promote Synaptic Protein Turnover by Endosomal MicroautophagyUytterhoeven et al.NeuronNovember 18, 2015In BriefUytterhoeven, Lauwers, and Maes et al. conducted a proteome-wide screen for membrane-deforming proteins and found the chaperone Hsc70-4. They show that Hsc70-4-dependent membrane deformation promotes endosomal microautophagy at presynaptic terminals, facilitating the degradation of synaptic proteins and promoting neurotransmitter release. Full-Text PDF Open Archive