Neuronale Zellbiologie & Synaptische Funktion

The presynaptic compartment

From sensory perception to learning and memory, the functioning of the nervous system is dependent upon communication between neurons. Dysfunction of neuronal signaling results in neurological and neurodegenerative disorders ranging from autism to epilepsy and Alzheimer's disease. During neurotransmission at synapses specialized 40 nm secretory organelles termed synaptic vesicles undergo calcium-regulated fusion at the presynaptic active zone to release neurotransmitters that are recognized by neurotransmitter receptors at the postsynaptic side.   

The Haucke lab aims to address three fundamental questions regarding the formation and maintenance of the presynaptic compartment:

1) How are key presynaptic components such as synaptic vesicles and active zone proteins formed in the neuronal soma, axonally transported, and assembled into nascent synapses? How do forming synapses communicate with the soma? Do similar mechanisms govern synapse formation and setting synaptic weight? 

2) How and how fast are synaptic vesicle membranes internalized post-fusion and how are synaptic vesicles then reformed to maintain neurotransmission?

3) How is a functional synapse adapted and maintained in the mature and aging nervous system to sustain and plastically modulate neurotransmission, e.g. via neuronal autophagy and the endolysosomal system?

To address these questions, we combine optical imaging, suprer-resolution light and correlative light and electron microscopy, live imaging, electrophysiology, and genetic approaches in primary mouse neurons and slice preparations and in human induced pluripotent stem cell derived neurons as models.

Formation of the presynaptic compartment

Our ability to move, to process sensory information or to form, store and retrieve memories crucially depends on the function of neuronal synapses. Synapses comprise a presynaptic compartment harboring the machinery for neurotransmitter release and an associated postsynaptic compartment that processes the neurotransmitter signal. During decades of research we have acquired a wealth of knowledge regarding the mechanisms of neurotransmitter release and information processing in the postsynaptic compartment. In great contrast, we know surprisingly little about the pathways that direct the formation, transport, and assembly of the complex molecular machines that make up a functional presynapse and that control synaptic weight in the mature CNS.

Recent work has focussed on testing the hypothesis that protein and organelle delivery play key roles in setting synaptic weight and in defining synaptic states. Two parallel work programs have been pursued: (1) The analysis of the mechanisms by which presynaptic material is delivered to nascent synapses in excitatory human neurons and (2) exploring the role of signaling mechanisms that may control the delivery pathway to set synaptic weight.

 To monitor the axonal transport and delivery of presynaptic components to nascent presynaptic compartments in developing mammalian central nervous system neurons we adopted a previously described protocol for the differentiation of human induced pluripotent stem cells (iPS) into glutamatergic neurons (iN) by Doxycycline-triggered expression of the transcription factor Ngn2. Co-culture of these developing iN with primary mouse astrocytes enabled the formation of functional pre- and postsynaptic compartments. Using this system we found that presynaptic biogenesis involves the axonal transport of precursor vesicles (PVs) harboring multiple newly synthesized presynaptic components including synaptic vesicle (SV) and active zone (AZ) proteins as well as the cell adhesion protein Neurexin1. Moreover, we could show using correlative light and electron microscopy that axonally transported PVs are distinct from conventional secretory organelles, recycling endosomes, and mature lysosomes and from SVs (Figure 1).

Instead, they may represent a neuron-specific biogenesis organelle, which derives from a pathway that sorts lysosomal membrane proteins. Further molecular mechanistic analysis by CRISPR-based genome engineering, lentiviral transduction, and biochemical approaches revealed that presynaptic biogenesis is mediated by Arl8A/B- and kinesin KIF1A-dependent axonal transport and delivery of vesicular and tubular PV carriers to nascent synapses. Consistently, these PVs displayed lipid hallmarks characteristic of the late endosomal/ lysosomal system. Similar findings were made in D. melanogaster motoneurons in studies conducted in collaboration with the Sigrist lab at FU Berlin (Vukoja et al., Neuron 2018).

We therefore postulate that PVs represent a neuron-specific biogenesis organelle, which may derive from a pathway that sorts lysosomal membrane proteins (Figure 2). We predict that this pathway is not only used in development but also in the mature nervous system to set and alter synaptic strength over time periods well beyond the classical paradigms of long-term plasticity.

Synaptic vesicle  exo- and endocytosis and synaptic vesicle reformation

One focus of the lab in the past has been the dissection of the mechanisms of presynaptic membrane retrieval and synaptic vesicle reformation (Figure 3). 

This has resulted in several major discoveries. We found that at physiological temperature SV internalization occurs on several time scales, from less than a second to several seconds and is mediated by dynamin, BAR domain proteins, and the actomyosin cytoskeleton and the activity of actin-nucleating formins such as mDia1 (Soykan et al., Neuron 2017) (Figure 3b). Surprisingly, this pathway of SV membrane endocytosis is independent of clathrin and its major adaptor protein 2 (AP-2), key essential proteins in clathrin-mediated endocytosis in non-neuronal cells. Clathrin/AP-2 instead appear to operate predominantly at internal endosome-like vacuoles to regenerate synaptic vesicles but, is dispensable for compensatory membrane retrieval. Loss of AP-2 in vivo causes the accumulation of endosome-like vacuoles and a depletion of synaptic vesicles, resulting in severely impaired neurotransmission and postnatal lethality (Figure 4).


These experimental results together with theoretical modelling provide a conceptual framework for how synapses capitalize on clathrin-independent membrane retrieval via dynamin/ F-actin and clathrin/ AP-2-mediated SV reformation from endosome-like vacuoles to maintain excitability over a broad range of stimulation frequencies (Kononenko et al., Neuron 2014; Kononenko & Haucke, Neuron 2015) (Figure 4). Consistent with this, we found that mutation of AP-2 causes developmental and epileptic encephalopathy in humans (Helbig et al., Am. J. Hum. Genet. 2019). Our studies highlight the importance of proper AP-2 function for SV recycling (Lopez-Hernandez et al., eLife 2022) and excitatory/ inhibitory balance in the central nervous system.

We have identified specific endocytic adaptors for SV protein sorting such as stonin 2, AP180, and CALM that control the fidelity of SV protein sorting (Kononenko et al., Proc. Natl. Acad. Sci.USA 2013; Koo et al., Neuron 2015). We further discovered using conditional knockout mice that CALM, a protein implicated in Alzheimer’s disease in humans, controls the surface levels of calcium-permeable (CP)-AMPA-type glutamate receptors (AMPARs) and, thereby, reciprocally regulates long-term potentiation (LTP) and long-term depression (LTD) in vivo to modulate learning. We demonstrated that CALM selectively facilitates the endocytosis of ubiquitinated CP-AMPARs via a mechanism that depends on ubiquitin recognition by its ANTH domain but is independent of clathrin. These data thus identify CALM and related ANTH domain–containing proteins as the core endocytic machinery that determines the surface levels of CP-AMPARs to bidirectionally control synaptic plasticity and modulate learning in the mammalian brain (Azarnia-Tehran et al. Sci. Adv.2022).

In a further set of studies, we have unravelled mechanisms that couple SV fusion and to the endocytic recycling of SV membranes. For example, we have found that loss of the endocytic proteins AP-2 or intersectin 1 in mice causes selective defects in fast neurotransmitter release that may be related to the clearance of release sites for neurotransmitter rather than defects in presynaptic membrane retrieval (Jung, Maritzen et al., EMBO J. 2015, Sakaba et al., Proc. Natl. Acad. Sci. USA 2013., Jaepel et al., Cell Reports 2020). Most recently, we discovered that the SV calcium sensor Synaptotagmin 1 couples exocytic SV fusion to the endocytic retrieval of SV membranes by promoting the local activity-dependent formation of the signaling lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at presynaptic sites. Interference with these mechanisms impairs PI(4,5)P2-triggered SV membrane retrieval but not exocytic SV fusion (Figure 5). These findings demonstrate that the coupling of SV exocytosis and endocytosis involves local Synaptotagmin 1-induced lipid signaling to maintain presynaptic membrane homeostasis in central nervous system neurons (Bolz et al. Neuron 2023).

Functions of neuronal autophagy and the endolysosomal system in neuronal health and neurotransmission

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 autophagy and the endolysosomal system contribute to neuronal and synaptic protein turnover, and what the main physiological substrates of autophagy are in healthy neurons remains poorly understood.

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 (Figure 6), resulting in increased excitatory neurotransmission and compromised postnatal viability in vivo. We found that the gain in excitatory neurotransmission in atg5 KO neurons is a consequence of elevated calcium release from ER stores via ryanodine receptors accumulated in axons and at presynaptic sites (Kuijpers et al., Neuron 2021). These findings suggets a model in which neuronal autophagy controls axonal ER calcium stores to regulate neurotransmission in healthy neurons and in the brain.

Present studies aim at addressing the mechanisms and components that regulate axonal ER-phagy in central nervous system neurons. These studies are of importance for the development of novel strategies to combat neurodegeneration and aging-associated memory decline.

Selected publications

  • Rizalar FS, Lucht MT, Petzoldt A, Kong S, Sun J, Vines JH, Telugu NS, Diecke S, Kaas T, Bullmann T, Schmied C, Löwe D, King JS, Cho W, Hallermann S, Puchkov D, Sigrist SJ, Haucke V. (2023) Phosphatidylinositol 3,5-bisphosphate facilitates axonal vesicle transport and presynapse assembly. Science 382(6667), 223-230. doi: 10.1126/science.adg1075. 
  • Bolz S, Kaempf N, Puchkov D, Krauss M, Russo G, Soykan T, Schmied C, Lehmann M, Müller R, Schultz C, Perrais D, Maritzen T, Haucke V. (2023) Synaptotagmin 1-triggered lipid signaling facilitates coupling of exo- and endocytosis. Neuron S0896-6273(23)00628-1.
  • Lopez- Hernandez, T., Takenaka, K., Mori, Y., Kongpracha, P., Nagamori, S., Haucke, V.*, Takamori, S.* (2022) Clathrin-independent endocytic retrieval of SV proteins mediated by the clathrin adaptor AP-2 at mammalian central synapses. eLife, 11:e71198. [*co-corresponding last authors]
  • Kuijpers, M., Kochlamazashvili, G., Stumpf, A., Puchkov, D., Swaminathan, A., Lucht, M.T., Krause, E., Schmitz, D., Haucke, V. (2021) Neuronal autophagy regulates presynaptic neurotransmission by controlling the axonal endoplasmic reticulum. Neuron 109, 299-313
  • Rizalar, F. S., Roosen, D., Haucke, V. (2021) A presynaptic perspective on transport and assembly mechanisms for synapse formation. Neuron 109, 27-41
  • Vukoja A., Rey, U., Petzoldt, A.G., Ott, C., Vollweiter, D., Quentin, C., Puchkov, D., Reynolds, E., Lehmann, M., Hohensee, S., Rosa, S., Lipowsky, R., Sigrist, S., Haucke, V. (2018) Presynaptic biogenesis requires axonal transport of lysosome-related vesicles. Neuron, 99, 1216-1232.
  • Soykan, T., Kaempf, N., Sakaba, T., Vollweiter, D., Goerdeler, F., Puchkov, D., Kononenko, N.L., Haucke, V. (2017) Synaptic vesicle endocytosis occurs on multiple timescales and is mediated by formin-dependent actin assembly. Neuron, 93, 854-866
  • Kononenko & Haucke (2015) Molecular mechanisms of presynaptic membrane retrieval and synaptic vesicle reformation. Neuron, 85, 484-496.
  • Kononenko, N.L., Puchkov, D., Classen, G.A., Walter, A., Pechstein, A., Sawade, L., Kaempf, N., Trimbuch, T., Lorenz, D., Rosenmund, C., Maritzen, M., Haucke, V. (2014) Clathrin/ AP-2 mediate synaptic vesicle reformation from endosome-like vacuoles but are not essential for membrane retrieval at central synapses. Neuron, 82, 981-988.
  • Haucke, V., Neher, E., Sigrist, S.J. (2011) Protein scaffolds in the coupling of synaptic exocytosis and endocytosis. Nat Rev. Mol. Neurosci., 12, 127-138.