Neuronal autophagy controls the axonal endoplasmic reticulum to regulate neurotransmission in healthy neurons

ABSTRACT

Neurons are long-lived cells that communicate via release of neurotransmitter at specialized contacts termed synapses. The maintenance of neuronal health and the regulation of synaptic function requires the efficient removal of damaged or dispensable proteins and organelles from synapses. How macroautophagy/autophagy contributes to neuronal and synaptic protein turnover, and what its main physiological substrates are in healthy neurons is largely unknown. We have now shown that loss of neuronal autophagy facilitates presynaptic neurotransmission by controlling the axonal endoplasmic reticulum and, thereby, axonal and synaptic calcium homeostasis.

Neurotransmission is a fundamental process that drives the transfer of information between neurons. It requires the controlled release of neurotransmitter by calcium-triggered synaptic vesicle (SV) fusion at the axon terminal, also known as the presynapse. Once released into the synaptic cleft, neurotransmitters interact with receptors on the membrane of the receiving (or postsynaptic) neuron. Synapses are exceptionally dynamic, and synaptic proteins have to be added and removed to scale up or scale down synaptic communication in response to neuronal activity. Local degradative pathways, including the proteasome, the endo-lysosomal system and autophagy, are essential for keeping synapses dynamic. In addition, aggregate-prone proteins have been linked to neurodegenerative disorders, and degradation pathways are therefore vital for a healthy brain. The autophagy-lysosomal degradation pathway has become a focus of research in synaptic biology, as it has become evident that autophagy plays a role in a variety of pre- and postsynaptic processes, such as synapse development and the regulation of synapse efficacy. Despite the physiological importance of autophagy for neuronal health and function, the physiological substrates of neuronal autophagy and the mechanisms by which defects in neuronal autophagy affect synaptic function remain largely unknown.

In our recent study [1], we used electrophysiological and cell biological approaches in conditional atg5 knockout mice to demonstrate that loss of neuronal autophagy enhances neurotransmission by facilitating the exocytic fusion of SVs with the presynaptic membrane as a result of altered calcium homeostasis. To identify autophagy substrates that could conceivably regulate neurotransmission, we conducted an unbiased proteomic analysis of protein degradation in autophagy-deficient atg5 conditional knockout and wild-type neurons. Surprisingly, we did not observe any changes in the steady-state levels or turnover of presynaptic exo- or endocytic proteins involved in neurotransmission in atg5 KO neurons. Instead, gene ontology analysis indicated that the majority of proteins with reduced turnover in the absence of ATG5-mediated autophagy were proteins localized to the tubular endoplasmic reticulum (ER). Strikingly, fluorescence and electron microscopy showed that tubular ER primarily accumulated in KO axons and at presynapses. Collectively, our data showed that axonal ER is a major substrate for neuronal autophagy in healthy unperturbed neurons. So how can axonal ER accumulation lead to facilitated neurotransmission? Axons largely contain smooth tubular ER, an organelle known to play roles in lipid biosynthesis and transport, e.g. via membrane contacts with other organelles, and as a calcium storage and release site. One of the most prominent roles of calcium in neurons is the triggering of neurotransmitter release. During action potential firing, high levels of calcium accumulate within presynaptic nerve terminals, which trigger SV fusion and, thereby, neurotransmitter release. Hence, we hypothesized that the accumulation of tubular ER in axons might cause alterations in axonal and/or presynaptic calcium homeostasis resulting in increased presynaptic neurotransmission. Calcium imaging experiments indeed revealed defects in axonal calcium homeostasis that appeared correlated with a reduced calcium content in the lumen of the ER. As a result, atg5 KO neurons were defective in restoring normal presynaptic calcium levels in response to activity-induced calcium influx through voltage-gated calcium channels. These data suggest that loss of neuronal autophagy causes defects in axonal calcium buffering. Mechanistically, this defect was shown to be a consequence of elevated release of calcium from axonal ER stores via ryanodine receptors (RYRs), large calcium release channels in ER membranes. Our collective data thus suggest a model where neuronal autophagy regulates neurotransmission by locally controlling the tubular ER and associated proteins to control axonal and presynaptic calcium homeostasis (Figure 1).

Figure 1.

Neuronal autophagy is highly compartmentalized, with autophagosomes that originate primarily from axons and synapses while degradative lysosomes are enriched in the neuronal somata. The axonal ER membrane and associated proteins are a major substrate of steady state neuronal autophagy. Autophagy-deficient atg5 KO neurons accumulate tubular ER in axons and synaptic terminals, causing elevated calcium release from the ryanodine receptors (RYR) on the ER surface, resulting in increased neurotransmission

As often, many intriguing aspects and unanswered questions remain for future investigations. At this point, we do not know yet if axonal ER degradation is a constitutive process, intimately linked to the formation of autophagosomes in distal axons, or if degradation is selective and/or regulated by specialized autophagy receptors. Autophagy is known not only to degrade entire organelles, but often displays substrate selectivity at the level of organellar subdomains or individual proteins. For the degradative turnover of the ER, a process referred to as reticulophagy, a growing set of receptors have been identified that may target specific ER subdomains. Interestingly, we found that while RYR levels are significantly increased in atg5 KO axons, other ER associated proteins (e.g., translocon components and other calcium channels) are unaffected, implying selectivity in neuronal ER degradation. How this selectivity is regulated, i.e., which reticulophagy receptors mediate this process, remains an open question.

A further intriguing question pertains to the compartmentalization of neuronal autophagy (Figure 1). Inhibition of autophagy in neurons leads to compartment-specific cargo buildup of the ER in axons but not in dendrites and of the autophagy receptor SQSTM1/p62 in neuronal somata [1]. Surprisingly also, loss of ATG5 in neurons does not affect autophagic turnover of mitochondria [1]. One possible explanation for these puzzling observations is that autophagosomes execute different functions in different neuronal compartments and/or under different conditions. In this context it is interesting to note that under conditions of genetic perturbation of the presynaptic active zone in the absence of giant protein BSN (bassoon), SV proteins appear to become degraded by autophagic turnover via ATG5. Finally, although our work has focused primarily on the presynapse, it would be interesting to analyze if local ER degradation, e.g., via specific reticulophagy receptors, affects postsynaptic function.

It is tempting to speculate that reticulophagy not only regulates neurotransmission but, conversely, is regulated by neuronal activity itself, thereby placing autophagy in a position to provide feedback on brain activity. How neuronal reticulophagy is affected by neuronal activity or other stimuli is not known yet. Addressing these issues will provide a deeper understanding of how neuronal autophagy is regulated and how compartment-specific degradation processes affect neuronal communication. Lastly, reticulophagy might be an important therapeutic target for neurological diseases, such as spastic paraplegias, in which ER morphology or function is affected.

Funding Statement

Our research was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) [HA 2686/13-1 to V.H.], the Neurocure Cluster of Excellence (DFG-Exc-257), the German Ministry of Science (BMBF; SMARTAGE, to V.H.) and a Marie Skłodowska-Curie Postdoctoral Fellowship from the European Union (655604-SYNPT, to M.K.).

Disclosure statement

No potential conflict of interest was reported by the authors.